Luciferase sequences utilizing infrared-emitting substrates to produce enhanced luminescence

ABSTRACT

Provided herein are isolated polynucleotide encoding modified click beetle luciferase polypeptides that have enhanced luminescence and longer wavelength near-infrared signals. The disclosure also relates to near-infrared bioluminescence systems that include said modified click beetle luciferase polypeptides and novel luciferin derivatives, as well as methods of using said modified click beetle luciferase polypeptides and bioluminescence systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/049,150, filed Sep. 11, 2014, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an isolated polynucleotide encodingclick beetle luciferase variant polypeptides that have enhancedluminescence and produce longer, near-infrared wavelength signals. Thepresent invention also relates to near-infrared bioluminescence systemsthat include said click beetle luciferase variant polypeptides andinfrared-emitting substrates and methods of using said click beetleluciferase variant polypeptides and near-infrared bioluminescencesystems.

BACKGROUND

Bioluminescence with a longer-wavelength and lower-energy emission is ofsignificant interest both for multiplexing applications with multipleemission colors and for in-depth tissue imaging where shorterwavelengths tend to be strongly absorbed. Many standard systems foroptical imaging have limited utility in a whole-animal context due tothe diminished transmission of light through biological samples. Lightpenetration is limited by the absorption coefficients of particularcomponents in blood. Strong absorption by Hemoglobin (Hb) and oxygenatedhemoglobin (HbO₂) diminish transmission and penetration depth of lightthrough blood and animal tissues. Luminescent systems that emit light inthe far-red and near-infrared region (680-900 nm) allow for optimalimaging due to the minimum absorbance spectrums of Hb and HbO₂. Thisregion of maximum light penetration is known as the whole animal“optical window.” Bioluminescent reporter systems have been usedextensively in research animals, yet still suffer from the limitationsof diminished tissue penetration. Typical bioluminescent light emissionwavelengths (460-620 nm) occur in a region with limited penetrationdepth. The ideal bioluminescent reporter systems in whole animals wouldbenefit greatly from the bright light emission in the region of 680-900nm. While numerous bioluminescent systems have been modified to shiftvisible light emission toward the red, none have achieved a strong redemission to overlap significantly with the critical “optical window” ofblood transmittance.

Previous approaches for molecular imaging in vivo include quantum dotconjugates that luminesce by bioluminescence resonance energy transfer(BRET) in the absence of external excitation. These conjugates, preparedby coupling carboxylate-presenting quantum dots to a mutant of thebioluminescent protein of Renilla reniformis luciferase, emit along-wavelength (from red to near-infrared) bioluminescent light incells and in animals, even in deep tissues. However, this approach islimited by the signal intensity for Renilla luciferase and by therelatively low aqueous solubility of the coelenterazine substrate.Another approach, e.g., Aka Lumine (Wako), utilizes a luciferinderivative which is dimmer and less red (675 nm) (see FIG. 19A and FIG.19B).

Therefore, there is a need in small animal optical imaging applicationsfor longer wavelength and lower energy-emitting near-infraredbioluminescence systems that will make it possible to detect signalsfrom deep tissue, where standard near-infrared bioluminescence systems(450-620 nm) tend to be strongly absorbed.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated polynucleotide encodinga click beetle red luciferase (CBR) variant polypeptide having at least80% amino acid sequence identity to SEQ ID NO: 1 and comprising at leastone amino acid substitution at a position corresponding to position 4,16, 34, 47, 51, 52, 55, 72, 73, 74, 79, 82, 83, 87, 89, 104, 109, 113,117, 119, 124, 130, 131, 133, 136, 144, 146, 156, 159, 170, 179, 186,200, 211, 218, 224, 225, 226, 228, 229, 234, 247, 251, 252, 253, 255,280, 281, 285, 308, 309, 310, 319, 329, 334, 335, 337, 346, 348, 349,350, 352, 354, 355, 358, 363, 370, 377, 390, 393, 394, 400, 401, 409,412, 420, 422, 431, 437, 439, 444, 445, 453, 455, 467, 471, 473, 479,484, 489, 496, 501, 503, 508, 516, 528, 531, 535, 537, 539, orcombination thereof, of SEQ ID NO: 1, wherein the variant CBRpolypeptide has at least one of enhanced luminescence, altered lightemission wavelength, altered substrate specificity, or a combinationthereof, as compared to a CBR polypeptide of SEQ ID NO: 1. The CBRvariant polypeptide may further comprise at least one amino acidsubstitution at a position corresponding to position 351, 389, 457, orcombination thereof, of SEQ ID NO: 1. The CBR variant polypeptide maycomprise a substitution corresponding to at least one of R4H, H16Q,H34Y, D47E, S51N, Y52C, F55L/V, K72E, I79V, M73K/T, N74S, E82G, N83H,F87S, I89V, V104D, I109N/V, L113Q, M117T, I119F/T, I124V, N130K,I131N/T, N133D, K136N, F144L, K146E, N156D, N156K, G159D, Y170C, K179S,V186A, G200G, N211N, H218L/Y, G225S, T226C/G/H/N/Q/Y, L228P, I229V,V234A, G251S, G251I, Y252C, V255D/F, E253K, R280S, S281N/Q, V285A,I309T, E319G, N329D, R334E/Q/H/S/N/K, C335S, K337E, I346N, Q348H/E,L350P, G351K/R, D352N, R355G, S358P, T363A/S, I370T, I389F/G/S/V, I390I,M393K/L, V394M, N400D, N401S, I409T, D412G, F420F, Y422C, V431A, E437G,I439V, S444C/R/T, Q445H, E453K, V455D, K457N, D471V, E473A, S479T,K484E/M/R, E489V, Y496H, E501G, V503M, Y508C, V516A, T528A, E531G,Q535H, L537W, K539R, or combinations thereof, or SEQ ID NO: 1. The CBRvariant polypeptide may comprise an amino acid substitution at aposition corresponding to position 389, 444, and 251 of SEQ ID NO: 1.The amino acid substitutions may comprise I389F, S444R, and G251S. TheCBR variant polypeptide may comprise an amino acid substitution atpositions corresponding to positions 334 and 351 of SEQ ID NO: 1. Theamino acid substitutions may comprise R334S and G351R. The CBR variantpolypeptide may further comprise an amino acid substitution at aposition corresponding to positions 51 and 444 of SEQ ID NO: 1. Theamino acid substitutions may comprise S51N and S444R. The CBR variantpolypeptide may comprise an amino acid polypeptide of SEQ ID NO: 2, SEQID NO: 3, or SEQ ID NO: 4. The CBR variant polypeptide may have enhancedluminescence compared to a CBR polypeptide of SEQ ID NO: 1. The CBRvariant polypeptide may have enhanced luminescence when a luciferin isutilized by the CBR variant polypeptide to generate luminescence. TheCBR variant polypeptide may have enhanced luminescence when a luciferinderivative is utilized by the CBR variant polypeptide to generateluminescence. The luciferin derivative may comprise:

The CBR variant polypeptide may have at least 2 fold increase inluminescence compared to a CBR polypeptide of SEQ ID NO: 1. The CBRvariant polypeptide may have at least 4 fold increase in luminescencecompared to a CBR polypeptide of SEQ ID NO: 1. The CBR variantpolypeptide may have altered light emission spectra compared to a CBRpolypeptide of SEQ ID NO: 1. The CBR variant polypeptide may be able toemit light at a longer wavelength when a luciferin is utilized by theCBR variant polypeptide to generate luminescence. The CBR variantpolypeptide may be able to emit light at a longer wavelength when aluciferin derivative is utilized by the CBR variant polypeptide togenerate luminescence. The luciferin derivative may comprise:

If the luciferin derivative comprises

the CBR variant polypeptide may emit light having a shift in spectralmaximum of at least about 1 nm to at least about 100 nm relative to thelight produced by the CBR polypeptide of SEQ ID NO: 1. The CBR variantpolypeptide may emit light having a spectral maximum between about 650nm to about 800 nm. The CBR variant polypeptide may emit light having aspectral maximum between about 725 nm to about 775 nm. The CBR variantpolypeptide may emit light having a spectral maximum of about 750 nm. Ifthe luciferin derivative comprises

the CBR variant polypeptide may emit light having a shift in spectralmaximum of at least about 1 nm to at least about 100 nm relative to thelight produced by the CBR polypeptide of SEQ ID NO: 1. The CBR variantpolypeptide may emit light having a shift in spectral maximum of atleast about 75 nm relative to the light produced by the CBR polypeptideof SEQ ID NO: 1. The CBR variant polypeptide may emit light having aspectral maximum between about 650 nm to about 800 nm. The CBR variantpolypeptide may emit light having a spectral maximum between about 700nm to about 775 nm. The CBR variant polypeptide may emit light having aspectral maximum of about 725 nm. The CBR variant polypeptide may havealtered substrate specificity compared to a CBR polypeptide of SEQ IDNO: 1. The CBR variant polypeptide may have a change in relativespecificity relative to the CBR variant polypeptide in the presence of aluciferin compared to a luciferin derivative. The CBR variantpolypeptide may have a change in relative specificity relative to theCBR variant polypeptide in the presence of a luciferin derivativecompared to a different luciferin derivative. The luciferin derivativemay comprise:

The variant CBR polypeptide may have luciferase activity. The variantCBR polypeptide may have a Km for PBI-4813 of at least about 0.01 μM toat least about 5.00 μM. The variant CBR polypeptide may have a Km forPBI-4813 of at least about 0.50 μM to at least about 3.00 μM. Thevariant CBR polypeptide may have a Km for PBI-4813 of at least about0.82 μM or 2.41 μM. The variant CBR polypeptide may have a Km forPBI-4739 of at least about 0.01 μM to at least about 5.00 μM. Thevariant CBR polypeptide may have a Km for PBI-4739 of at least about1.50 μM to at least about 4.50 μM. The variant CBR polypeptide may havea Km for PBI-4739 of at least about 2.33 μM or 3.95 μM. The variant CBRpolypeptide may have a relative Vmax that is at least 2 fold higher thanthe relative Vmax of a CBR polypeptide of SEQ ID NO: 1 using PBI-4813 asa substrate. The variant CBR polypeptide may have a relative Vmax thatis at least 2 fold higher than the relative Vmax of a CBR polypeptide ofSEQ ID NO: 1 using PBI-4739 as a substrate. The sequence may have beencodon-optimized. The sequence may comprise a polynucleotide of SEQ IDNOs: 6-9. The polynucleotide may further encode a polypeptide ofinterest linked to the CBR variant polypeptide, the polypeptide ofinterest and the CBR variant polypeptide capable of being expressed as afusion protein. The polypeptide of interest may comprise HALOTAG®.

The present invention is directed to a vector comprising thepolynucleotide, or a fragment thereof, as described above. Thepolynucleotide may be operably linked to a promoter.

The present invention is directed to a cell comprising thepolynucleotide as described above or the vector as described above.

The present invention is directed to a non-human transgenic animalcomprising the cell as described above.

The present invention is directed to a non-human transgenic animalcomprising the polynucleotide as described above or the vector asdescribed above.

The present invention is directed to a CBR variant polypeptide encodedby the polynucleotide as described above.

The present invention is directed to a circularly permuted luciferasecomprising the polypeptide encoded by the polynucleotide as describedabove or a fragment thereof.

The present invention is directed to a fusion protein comprising a CBRvariant polypeptide encoded by the polynucleotide as described above.

The present invention is directed to a near-infrared bioluminescencesystem comprising the polynucleotide as described above and a luciferinderivative. The luciferin derivative may comprise:

The present invention is directed to a method of producing a CBR variantpolypeptide comprising growing the cell as described above underconditions that permit expression of the CBR variant polypeptide.

The present invention is directed to a method of producing a CBR variantpolypeptide comprising introducing the vector as described above into acell under conditions which permit expression of the CBR variantpolypeptide.

The present invention is directed to a kit comprising the polynucleotideas described above or the vector as described above.

The present invention is directed to a kit comprising the CBR variantpolypeptide as described above. The kit may further comprise at leastone of:

(a)

and(b) a buffer reagent.

The present invention is directed to a bioluminescence resonance energytransfer (BRET) system comprising: a first fusion protein including afirst target protein and a bioluminescence donor molecule, wherein thebioluminescence donor molecule is a CBR variant encoded by thepolynucleotide as described above; a second fusion protein including asecond target protein and a fluorescent acceptor molecule; and a CBRsubstrate. The CBR substrate may be a luciferin or luciferin derivative.The luciferin derivative may comprise:

The present invention is directed to a method for measuringbioluminescence using at least one of the polynucleotide as describedabove; the vector as described above; the cell as described above; theanimal as described above; the CBR variant polypeptide as describedabove; the circularly permuted luciferase as described above; the fusionprotein as described above, or the near-infrared bioluminescence systemas described above. The bioluminescence may be measured in a live,intact non-human animal.

The present invention is directed to a method of measuring the enzymaticactivity of a luminogenic protein. The method comprises contacting aluminogenic protein, a deprotecting enzyme, and a protected luminophore;and detecting light produced from the composition, wherein theluminogenic protein is a CBR variant encoded by the polynucleotide asdescribed above and the luminophore is a luciferin derivative comprising

The enzymatic activity may be measured in a live, intact non-humananimal.

The present invention is directed to a method for measuring the activityof a non-luminescent enzyme of interest. The method comprises (a)providing a luminogenic molecule wherein the molecule is a substrate forthe non-luminescent enzyme of interest and a pro-substrate of a CBRvariant encoded by the polynucleotide as described above; (b) contactingthe luminogenic molecule with at least one non-luminescent enzyme ofinterest and at least one CBR variant to produce a reaction mixture; and(c) determining activity of the non-luminescent enzyme of interest bymeasuring luminescence of the reaction mixture. The luminogenic moleculemay be a modification of

The non-luminescent enzyme of interest may be a protease enzyme, acytochrome P450 enzyme, a monoamine oxidase enzyme, or a glutathioneS-transferase enzyme. The activity of the non-luminescent enzyme may bemeasured in a live, intact animal.

The present invention is directed to a method to detect the presence ofat least two molecules in a sample or a cell. The method comprisescontacting the sample or cell with a first reporter molecule comprisinga CBR variant encoded by the polynucleotide as described above, whereinthe first reporter molecule is operatively linked to a first componentof the sample or cell; contacting the sample with a second reportermolecule, wherein the second reporter molecule is operatively linked toa second component of the sample or cell; and detecting the presence ofthe first and second reporter molecules to determine the presence and/oramounts of the first and second components in the sample or cell.

The present invention is directed to a method to detect an interactionbetween a first protein and a second protein in a sample. The methodcomprises contacting a sample with:

wherein the sample comprises: a first polynucleotide encoding a firstfusion protein, wherein the first fusion protein comprises a firstfragment of a luminescent enzyme and a first protein; and a secondpolynucleotide encoding a second fusion protein, wherein the secondfusion protein comprises a second fragment of the luminescent enzyme anda second protein; and detecting luminescence in the sample. Thedetection of luminescence indicates an interaction between the firstprotein and the second protein, wherein the luminescent enzyme isencoded by the isolated polynucleotide as described above. When thefirst protein and second protein interact, the first fragment of theluminescent enzyme and the second fragment of the luminescent enzyme mayreconstitute a full-length enzyme capable of stably binding thecell-permeable substrate.

The present invention is directed to a method to detect an interactionbetween a first protein and a second protein in a sample. The methodcomprises contacting a sample with:

wherein the sample comprises: a first polynucleotide encoding a firstfusion protein, wherein the first fusion protein comprises a luminescentenzyme and a first protein, wherein the luminescent enzyme is encoded bythe isolated polynucleotide of any one of claims 1-46; and a secondpolynucleotide encoding a second fusion protein, wherein the secondfusion protein comprises a fluorescent acceptor molecule and a secondprotein; and (b) detecting bioluminescence resonance energy transfer(BRET) in the sample, indicating an interaction or close proximity ofthe bioluminescent donor and the fluorescence acceptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chemical structure of PBI-4739 and PBI-4813.

FIG. 2A and FIG. 2B illustrate spectral scans of click beetle redluciferase (CBR) using PBI-4813 (FIG. 2A) and PBI-4739 (FIG. 2B) assubstrates.

FIG. 3A and FIG. 3B illustrate spectral scans of Ultra-Glo™ Luciferaseusing PBI-4813 (FIG. 3A) and PBI-4739 (FIG. 3B) as substrates.

FIG. 4A and FIG. 4B illustrate spectral scans of QuantiLum® RecombinantLuciferase (“Firefly”) using PBI-4813 (FIG. 4A) and PBI-4739 (FIG. 4B)as substrates.

FIG. 5 illustrates the normalized luminescence of CBR variants and CBR(“WT”) using PBI-4813 (dark bars) and PBI-4739 (light bars) assubstrates.

FIG. 6 illustrates the normalized luminescence of CBR variants and CBRin HEK293 cells using PBI-4813 (dark bars) and PBI-4739 (light bars) assubstrates.

FIG. 7 illustrates the normalized luminescence of CBR variants and CBRin HeLa cells using PBI-4813 (dark bars) and PBI-4739 (light bars) assubstrates.

FIG. 8 illustrates the normalized luminescence of CBR variants and CBR(“WT”) using PBI-4813 (dark bars) and PBI-4739 (light bars) assubstrates.

FIG. 9 illustrates the normalized luminescence of CBR variants and CBR(“ATG 685”) using luciferin (“D-LH2”; dark bars), PBI-4813 (medium darkbars) and PBI-4739 (light bars) as substrates.

FIG. 10 illustrates the normalized luminescence of CBR variants ATG 1230and ATG 1250 and CBR using PBI-4813 (dark bars) and PBI-4739 (lightbars) as substrates.

FIG. 11A and FIG. 11B illustrate the spectral properties of CBR variantsATG 1230 and ATG 1240 compared with CBR using PBI-4813 (FIG. 11A) andPBI-4739 (FIG. 11B) as substrates.

FIG. 12A and FIG. 12B illustrate the Km titration of CBR variants ATG1230 and ATG 1240 compared with CBR for PBI-4813 (FIG. 12A) and PBI-4739(FIG. 12B) as substrates.

FIG. 13 illustrates the kinetic profile of CBR and CBR variants ATG 1230and ATG 1240 in live cell at 37° C. using PBI-4813 and PBI-4739 assubstrates.

FIG. 14 illustrates the luminescence between CBR variants ATG 1230 andATG 1240 compared with CBR using PBI-4813 (medium dark bars) andPBI-4739 (light bars) as substrates and with firefly luciferase (“Luc2”)using luciferin (“D-LH2”) as substrate (dark bar).

FIG. 15 illustrates the normalized luminescence of CBR variants usingluciferin (“D-LH2”; light bars), PBI-4813 (dark bars), and PBI-4739(medium dark bars) as substrates.

FIG. 16A and FIG. 16B illustrate live cell substrate titration of CBRvariant ATG 1240 compared to CBR using PBI-4739 (FIG. 16A) and PBI-4813(FIG. 16B) as substrates.

FIG. 17 illustrates the luminescence measured using long pass filters tomimic tissue attenuation of CBR variant ATG 1240 using luciferin(“D-LH2”; medium dark bars), PBI-4813 (medium light bars), and PBI-4739(light bars) as substrates compared with firefly luciferase (“Luc2”)using luciferin (dark bars).

FIG. 18 illustrates the amino acid sequence of CBR with target aminoacid sites for insertion mutagenesis bolded and underlined.

FIG. 19A and FIG. 19B illustrate the luminescence of CBR using AkaLumine, PBI-4739 and PBI-4813 (FIG. 19A) and the spectral scan ofQuantiLum® Recombinant Luciferase using Aka Lumine as substrate (FIG.19B).

FIG. 20A and FIG. 20B illustrate the luminescence of codon optimized CBRvariants expressed in CHO cells (FIG. 20A) or 3T3 cells (FIG. 20B) usingBright-Glo™ assay buffer.

FIG. 21A and FIG. 21B illustrate the Km titration of CBR for PBI-4813and D-luciferin (“D-LH2”) as substrates in live HeLa cells.

FIG. 22 illustrates the Km titration of CBR for PBI-4813, PBI-4739, andD-luciferin as substrates in live HEK293T cells.

FIG. 23A and FIG. 23B illustrate the Km titration of CBR variant ATG1230 compared with CBR for PBI-4813 (FIG. 23A) and PBI-4739 (FIG. 23B)as substrates in live HEK293T cells.

DETAILED DESCRIPTION

Disclosed herein are click beetle red luciferase (CBR) variants thathave enhanced performance in the near-infrared (near-IR) range usingluciferin and/or novel luciferin derivatives. The disclosed CBR variantshave enhanced performance in vitro and in living cells, e.g., increasedbrightness and longer wavelengths. Also disclosed herein are near-IRbioluminescence systems that comprise the CBR variants and novelluciferin derivatives. The disclosed near-IR bioluminescence systemshave enhanced performance in the near-IR range and address sensitivitylimitations that are related to tissue absorption and associated withother bioluminescent detections systems. Unlike other bioluminescencesystems, such as VivoGlo™ (Promega Corp.), the disclosed near-IRbioluminescence systems provide near-IR luminescence signals thatpenetrate tissue well and provide enhanced detection of signals comingfrom deep animal tissues. The present disclosure also encompassesmethods of using and kits including the CBR variants and/orbioluminescence systems.

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting. As used in the specification and theappended claims, the singular forms “a,” “and” and “the” include pluralreferences unless the context clearly dictates otherwise. The presentdisclosure also contemplates other embodiments “comprising,” “consistingof” and “consisting essentially of,” the embodiments or elementspresented herein, whether explicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

As used herein, “bioluminescence” or “luminescence” is light produced asa result of a reaction between an enzyme and a substrate that generateslight.

In general, “enhanced” means that the particular property (e.g.bioluminescence or luminescence) is increased relative to that of areference luciferase plus luciferin combination or luciferase underconsideration, where the increase is at least 1%, at least 2%, at least3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 25%,at least 50%, at least 75%, at least 90%, at least 100%, at least 200%,at least 500%, or at least 1000% greater than the reference luciferaseplus luciferin combination or luciferase under consideration.

The term “luciferin substrate” as used herein refers to a moleculecapable of creating light via a chemical or biochemical reaction (e.g.,luciferin, luciferin derivative, or a functional analog thereof).Suitable luciferin substrates for luciferase enzymes include luciferin,luciferin derivatives, and functional analogs of luciferins. In someembodiments, functional analogs of luciferins include modifiedluciferins including derivatives of these compounds. Exemplary compoundsinclude those disclosed in U.S. patent application Ser. No. 14/200,563.

The naturally-occurring substrate for beetle luciferases is fireflyluciferin, a polytherocyclic organic acid,D-(−)-2-(6′-hydroxy-2′-benzoth-iazolyl)-Δ²-thiazolin-4-carboxylic acid(“D-luciferin” also referred to as “luciferin”). Luciferin may beisolated from nature (e.g., from fireflies) or synthesized. Syntheticluciferin can have the same structure as the naturally occurringluciferin or can be derivatized, so long as it functions analogously.Examples of derivatives of luciferin include D-luciferin methyl esterand other esters of luciferase that are hydrolyzed or acted upon byesterases in a sample to yield luciferin, and naphthyl- andquinolyl-luciferin (Branchini et al., 1989). There are multiplecommercial sources for luciferin (e.g., Promega Corp. Madison, Wis.).

The term “luciferin derivative” as used herein refers to a type ofluminogenic molecule or compound having a substantial structure ofD-luciferin and is a luciferase substrate, e.g., aminoluciferin, orluciferase substrates disclosed in U.S. Patent Publication No.2007/0015790.

A “luciferase reaction mixture” contains a luciferase enzyme andmaterials that will allow the luciferase enzyme to generate a lightsignal. The materials needed, and the particular concentrations and/oramounts, of the materials needed to generate a luminescent signal willvary depending on the luciferase enzyme used as well as the type ofluciferase-based assay being performed. In general, for click beetleluciferase, including the CBR variants, these materials can include:ATP, magnesium (Mg2+) salt, such as magnesium sulfate, a click beetleluciferase enzyme, and a luciferin or novel luciferin derivative capableof generating light when the luciferin or novel luciferin derivative isused as a substrate for the click beetle luciferase. Often othermaterials will be added to the solution including: a buffer to maintainthe reaction at the proper pH, an additive such as PRIONEX or Bovineserum albumin (BSA) to help maintain luciferase activity, reducingagents, detergents, esterases, salts, amino acids, e.g. D-cysteine, etc.An example luciferase reaction mixture would contain a click beetleluciferase, MgSO₄, ATP, Tergitol NP-9, and Tricine.

A “luciferase detection mixture” contains materials that will allow forthe detection of a luciferase enzyme. The materials needed, and theparticular concentrations and/or amounts, of the materials needed togenerate a luminescent signal will vary depending on the luciferaseenzyme used as well as the type of luciferase-based assay beingperformed. In general, for click beetle luciferase, including the CBRvariants disclosed herein, these materials can include: ATP, magnesium(Mg2+) salt, such as magnesium sulfate, and a luciferase substrate,e.g., luciferin, luciferin derivative, functional analog, or novelluciferin derivative capable of generating light when the luciferasesubstrate, e.g., luciferin, luciferin derivative, functional analog, ornovel luciferin derivative is used as a substrate for the click beetleluciferase. Often other materials will be added to the solutionincluding: a buffer to maintain the reaction at the proper pH, anadditive such as PRIONEX or Bovine serum albumin (BSA) to help maintainluciferase activity, reducing agents, detergents, esterases, salts,amino acids, e.g. D-cysteine, etc. An example luciferase detectionmixture would contain a luciferase substrate, MgSO₄, ATP, Tergitol NP-9,and Tricine.

The term “luminescence” refers to the light output of the luciferase,e.g., the CBR variant, under appropriate conditions, e.g. in thepresence of a suitable substrate such as a luciferin or novel luciferinderivative. The light output may be measured as an instantaneous ornear-instantaneous measure of light output (which is sometimes referredto as “T=0” luminescence or “flash”) at the start of the luminescencereaction, which may be initiated upon addition of the luciferin or novelluciferin derivative substrate. The luminescence reaction in variousembodiments is carried out in a solution. In other embodiments, theluminescence reaction is carried out on a solid support. The solutionmay contain a lysate, for example from the cells in a prokaryotic oreukaryotic expression system. In other embodiments, expression occurs ina cell-free system, or the luciferase protein is secreted into anextracellular medium, such that, in the latter case, it is not necessaryto produce a lysate. In some embodiments, the reaction is started byinjecting appropriate materials, e.g. luciferin substrate, buffer, etc.,into a reaction chamber (e.g. a well of a multiwell plate such as a96-well plate) containing the luminescent protein. In still otherembodiments, the luciferase substrate is introduced into a host, whichmay be expressing a luciferase, e.g., a CBR variant, and measurements ofluminescence are made on the host or a portion thereof, which caninclude a whole organism or cells, tissues, explants, or extractsthereof. The reaction chamber may be situated in a reading device whichcan measure the light output, e.g. using a luminometer orphotomultiplier. The light output or luminescence may also be measuredover time, for example in the same reaction chamber for a period ofseconds, minutes, hours, etc. The light output or luminescence may bereported as the average over time, the half-life of decay of signal, thesum of the signal over a period of time, or the peak output.Luminescence may be measured in Relative Light Units (RLUs).

“Relative substrate specificity” is determined by dividing theluminescence of a luciferase in the presence of a test luciferinsubstrate by the luminescence of the luciferase in the presence of areference luciferin substrate. For example, relative specificity may bedetermined by dividing the luminescence of a luciferase with a novelluciferin derivative by the luminescence of the luciferase with adifferent luciferin (e.g. D-luciferin or novel luciferin derivative).The test luciferin substrate and the reference luciferin substrate thatare compared are considered a comparison substrate pair for determiningrelative substrate specificity.

A “change in relative substrate specificity” is determined by dividingthe relative substrate specificity of a test luciferase using acomparison substrate pair by the relative substrate specificity of areference luciferase using the same comparison substrate pair. Forexample, a change in relative specificity may be determined by dividingthe relative substrate specificity of a test luciferase with a novelluciferin derivative compared to a different luciferin (e.g. D-luciferinor novel luciferin derivative), by the relative substrate specificity ofa reference luciferase with the same novel luciferin derivative comparedto the same different luciferin used for the test luciferase.

The term “fusion polypeptide” or “fusion protein” refers to a chimericprotein containing a reference protein (e.g., CBR variant) joined at theN- and/or C-terminus to one or more heterologous sequences (e.g., anon-CBR polypeptide).

The term “identity,” in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same when compared and aligned for maximumcorrespondence over a comparison window or designated region as measuredusing any number of sequence comparison algorithms or by manualalignment and visual inspection. Methods of alignment of sequence forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted by the algorithm of Smith et al., (J. Mol.Biol. 147:195-197 (1981)), by the homology alignment algorithm ofNeedleman and Wunsch, (J. Mol. Biol., 48:443-453 (1970)), by the searchfor similarity method of Pearson and Lipman, (Proc. Natl. Acad. Sci.USA, 85:2444-2448 (1988)), by computerized implementations of algorithmse.g., FASTA, SSEARCH, GGSEARCH (available at the University of VirginiaFASTA server by William R. Pearsonhttp://fasta.bioch.virginia.edu/fasta_www2/fasta_intro.shtml), theClustal series of programs (Chenna et al., Nucl. Acids Res.31(13):3497-3500 (2003); available examples at http://www.ebi.ac.uk orhttp://www.ch.embnet.org), or other sequence analysis software. It isknown in the art that generating alignments with maximum correspondencebetween polypeptide sequences with significant sequence alterations(e.g. altered domain order, missing/added domains, repeated domains,shuffled domains, circular permutation) may involve the use ofspecialized methods, such as the ABA method (Raphael et al., Genome Res.14(11):2336-2346 (2004)), other suitable methods, or performing thealignment with two concatenated identical copies of the polypeptidesequences.

Nucleic acids are known to contain different types of “mutations”, whichrefers to an alteration in the sequence of a nucleotide at a particularbase position relative to the wild-type sequence. Mutations may alsorefer to insertion or deletion of one or more bases so that the nucleicacid sequence differs from a reference, e.g., a wild-type sequence, orreplacement with a stop codon. A “substitution” refers to a change in anamino acid at a particular position in a sequence.

The term “nucleic acid molecule,” “polynucleotide” or “nucleic acidsequence” as used herein, refers to nucleic acid, including DNA or RNA,that comprises coding sequences necessary for the production of apolypeptide or protein precursor. The encoded polypeptide may be afull-length polypeptide, a fragment thereof (less than full-length), ora fusion of either the full-length polypeptide or fragment thereof withanother polypeptide, yielding a fusion polypeptide.

A polynucleotide encoding a protein or polypeptide means a nucleic acidsequence comprising the coding region of a gene, or in other words, thenucleic acid sequence encoding a gene product. The coding region may bepresent in a cDNA, genomic DNA or RNA form. When present in a DNA form,the oligonucleotide may be single stranded (e.g., the sense strand) ordouble stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene, if needed, to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Other control or regulatory elements include, but are notlimited to, transcription factor binding sites, splicing signals,polyadenylation signals, termination signals and enhancer elements.

By “peptide,” “protein” and “polypeptide” is meant amino acid chains ofvarying lengths, regardless of post-translational modification (e.g.,glycosylation or phosphorylation). The nucleic acid molecules of theinvention encode a variant of a man-made (i.e. synthetic) variantprotein or polypeptide fragment thereof, which has an amino acidsequence that is at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity tothe amino acid sequence of the parental protein from which it isderived, where the parental protein can be a naturally-occurring (nativeor wild-type) sequence or a variant sequence which is subsequentlymodified further.

As used herein, “pure” or “purified” means an object species is thepredominant species present (i.e., on a molar and/or mass basis, it ismore abundant than any other individual species, apart from water,solvents, buffers, or other common components of an aqueous system inthe composition), and, in some embodiments, a purified fraction is acomposition wherein the object species comprises at least about 50% (ona molar basis) of all macromolecular species present. Generally, a“substantially pure” composition will comprise more than about 80% ofall macromolecular species present in the composition, in someembodiments more than about 85%, more than about 90%, more than about95%, or more than about 99%. In some embodiments, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “variant” refers to a modified version of a startingpolypeptide or polynucleotide sequence. The term “parental” is arelative term that refers to a starting sequence which is then modified.The parental sequence is generally used as a reference for the proteinencoded by the resulting modified sequence, e.g. to compare the activitylevels or other properties of the proteins encoded by the parental andthe modified sequences. The starting sequence can be anaturally-occurring (i.e. native or wild-type) sequence. The startingsequence can also be a variant sequence which is then further modified.A polypeptide sequence is “modified” when one or more amino acids (whichmay be naturally-occurring or synthetic) are substituted, deleted,and/or added at the beginning, middle, i.e., at an internal position,and/or end of the sequence. A polynucleotide sequence is “modified” whenone or more nucleotides are substituted, deleted, and/or added at thebeginning, middle, i.e., at an internal position, and/or end of thesequence, but which may or may not alter the amino acid encoded by thesequence. In some embodiments, the modifications produce a variant thatis a functional fragment of CBR or a particular CBR variant. Afunctional fragment is a fragment which is less than a full-lengthparental sequence which has the same functional activity as thefull-length parental sequence. Functional activity is the ability toexhibit luminescence. In some embodiments, the modifications produce avariant that is a permuted sequence of the parental sequence, such as acircularly permuted sequence and permuted sequences comprising deletionsand/or insertions.

Variant is also used herein to describe a protein with an amino acidsequence that is substantially identical to a referenced protein with anamino acid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree, and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function.

Substitution of amino acids having similar hydrophilicity values canresult in peptides retaining biological activity as is understood in theart. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. “Variant” also can be used to describe apolypeptide or a fragment thereof that has been differentiallyprocessed, such as by proteolysis, phosphorylation, or otherpost-translational modification, yet retains its biological activity.

The term “vector” refers to nucleic acid molecules into which fragmentsof DNA may be inserted or cloned and can be used to transfer DNAsegment(s) into a cell and capable of replication in a cell. Vectors maybe derived from plasmids, bacteriophages, viruses, cosmids, and thelike.

The term “wild-type” or “native” as used herein refers to a gene or geneproduct that has the characteristics of that gene or gene productisolated from a naturally occurring source. A wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designated the “wild-type” form of the gene. In contrast,the term “mutant” refers to a gene or gene product that displaysmodifications in sequence and/or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

2. CBR VARIANTS

The present disclosure provides variants of click beetle red luciferase(CBR) that utilize luciferin and luciferin derivatives and producelonger near-IR (NIR) wavelength signals. Various techniques describedherein were used to evolve CBR to produce brighter light output withextended conjugation luciferins and to identify sites for amino acidsubstitution to produce an improved synthetic CBR polypeptide. It wasfound that making one or more amino acid substitutions, either alone orin various combinations, produced synthetic CBR-type polypeptides havingenhanced luminescence (e.g., enhanced brightness) and longer wavelengthemissions. Also provided is a polynucleotide encoding a CBR variant ofthe invention or a fusion thereof, an isolated host cell having thepolynucleotide or the CBR variant or a fusion thereof, and methods ofusing the polynucleotide, CBR variant or a fusion thereof or host cellof the invention.

The CBR variants have at least one amino acid substitution at a positioncorresponding to position 4, 16, 34, 47, 51, 52, 55, 72, 73, 74, 79, 82,83, 87, 89, 104, 109, 113, 117, 119, 124, 130, 131, 133, 136, 144, 146,156, 159, 170, 179, 186, 200, 211, 218, 224, 225, 226, 228, 229, 234,247, 251, 252, 253, 255, 280, 281, 285, 308, 309, 310, 319, 329, 334,335, 337, 346, 348, 349, 350, 352, 354, 355, 358, 363, 370, 377, 390,393, 394, 400, 401, 409, 412, 420, 422, 431, 437, 439, 444, 445, 453,455, 467, 471, 473, 479, 484, 489, 496, 501, 503, 508, 516, 528, 531,535, 537, 539, or a combination thereof, of SEQ ID NO: 1. The CBRvariants may further include at least one amino acid substitution at aposition corresponding to position 351, 389, 457, or a combinationthereof, of SEQ ID NO: 1. The CBR variants may have at least one aminoacid substitution of corresponding to at least one of R4H, H16Q, H34Y,D47E, S51N, Y52C, F55L/V, K72E, I79V, M73K/T, N74S, E82G, N83H, F87S,I89V, V104D, I109N/V, L113Q, M117T, I119F/T, I124V, N130K, I131N/T,N133D, K136N, F144L, K146E, N156D, N156K, G159D, Y170C, K179S, V186A,G200G, N211N, H218L/Y, G225S, T226C/G/H/N/Q/Y, L228P, I229V, V234A,G251S, G251I, Y252C, V255D/F, E253K, R280S, S281N/Q, V285A, I309T,E319G, N329D, R334E/Q/H/S/N/K, C335S, K337E, I346N, Q348H/E, L350P,G351K/R, D352N, R355G, S358P, T363A/S, I370T, I389F/G/S/V, I390I,M393K/L, V394M, N400D, N401S, I409T, D412G, F420F, Y422C, V431A, E437G,I439V, S444C/R/T, Q445H, E453K, V455D, K457N, D471V, E473A, S479T,K484E/M/R, E489V, Y496H, E501G, V503M, Y508C, V516A, T528A, E531G,Q535H, L537W, K539R, or combinations thereof, of SEQ ID NO: 1. The CBRvariant polypeptide may have an amino acid substitution at a positioncorresponding to positions 389, 444, and 251 of SEQ ID NO: 1. The aminoacid substitutions may include I389F, S444R, and G251S. The CBR variantpolypeptide may have an amino acid substitution at a positioncorresponding to positions 334 and 351 of SEQ ID NO: 1. The amino acidsubstitutions may include R334S and G351R. The CBR variant may comprisean amino acid polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3.

Several of the CBR variants and parental CBR disclosed herein have beenassigned shorthand names to facilitate discussion. The term “ATG 343”(also referred to as “343” and “pF4Ag-CBR”) refers to a CBR polypeptidesequence of SEQ ID NO: 1. The CBR polypeptide sequence of SEQ ID NO: 1is encoded by the polynucleotide sequence of SEQ ID NO: 5. The term “ATG685” (also referred to as “685” and “pF4Ag-HT7-CBR”) refers to a CBRpolypeptide sequence of SEQ ID NO: 1 fused to HALOTAG®. The term “ATG1230” (SEQ ID NO: 3) refers to a CBR variant having the amino acidsubstitutions I389F, S444R, and G251S relative to SEQ ID NO: 1 (wherethe format “x#y” indicates a parent amino acid ‘x’ at a position ‘#’that is changed to variant amino acid ‘y’). The term “ATG 1240” (SEQ IDNO: 2) refers to a CBR variant having the amino acid substitutions R334Sand G351R relative to SEQ ID NO: 1. The term “ATG 1240+S51N+S444R” (SEQID NO: 4) refers to a CBR variant having the amino acid substitutions ofATG 1240, i.e., R334S and G351R of SEQ ID NO: 1, as well as amino acidsubstitutions S51N and S444R relative to SEQ ID NO: 1.

The sequences of a CBR variant are substantially the same as the aminoacid sequence of a corresponding parental CBR, e.g., SEQ ID NO: 1. Apolypeptide or peptide having substantially the same sequence means thatan amino acid sequence is largely, but is not entirely, the same andretains the functional activity of the sequence to which it is related.In general, two amino acid sequences are substantially the same if theyare at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99%, but less than 100%, amino acid sequence identity. Insome embodiments, the CBR variant is encoded by a recombinantpolynucleotide.

In some embodiments, the CBR variant has at least 60%, e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, aminoacid sequence identity to SEQ ID NOs: 1, 2, 3, or 4. Ordinarily, variantfragments are at least about 50 amino acids in length, often at leastabout 60 amino acids in length, more often at least about 70, 80, 90,100, 150, 200, 300, 400, 500 or 550 amino acids in length, or more andretain the ability to generate luminescence. A full length luciferase,fragment thereof, or variant thereof may be fused to heterologous aminoacid sequences and still be functional in the invention.

These CBR variants display at least one of: enhanced luminescence(including increased brightness, enhanced signal stability, signalduration, and/or decreased sensitivity to substrate inhibition); alteredlight emission spectra; and altered substrate specificity (i.e., changein relative substrate specificity). In various embodiments, the presentinvention encompasses novel luciferases that are present in solution assoluble proteins, chemically linked to other molecules (e.g., fusionproteins), or attached onto a solid surface (e.g., particles,capillaries, or assay tubes or plates).

a. Enhanced Luminescence

The enhanced luminescence of a CBR variant may be due to one or more ofthe following characteristics: enhanced light output (i.e. brightness),enhanced substrate specificity, enhanced signal stability, enhancedsignal duration, and/or decreased sensitivity to substrate inhibition.Enhanced signal stability includes an increase in how long the signalfrom a luciferase continues to luminesce, for example, as measured bythe half-life of decay of the signal in a time-course. The term“substrate inhibition” as used herein refers to the inhibition ofluciferase enzyme activity (e.g., inhibition of generating luminescenceand lower RLU values) at high concentration levels of substrate. A CBRvariant having decreased sensitivity to substrate inhibition does nothave lower RLU values with increasing amounts of substrate. For example,PBI-4813 at concentrations greater than ten times the Km inhibits theactivity of CBR and ATG1230, thus the luminescence generated, i.e., RLUvalues, is decreased, whereas ATG1240 does not show this inhibition withPBI-4813 and has decreased sensitivity to substrate inhibition.

Enhanced luminescence may be determined relative to the comparableproperty of a luciferase such as wild-type CBR, a CBR variant protein,Renilla luciferase (e.g., hRluc), or firefly luciferase (e.g., Luc2;luciferase from Photinus pyralis) combined with a native, known, ornovel substrate, as shown in the Examples below. For example, theluminescence of a given CBR variant in combination with a particularluciferin (including native, known, or novel luciferin, or derivativesthereof) may be compared to the properties of CBR combined with any of anative, known, or novel luciferins or derivatives thereof disclosedherein, using one or more of the assays disclosed in the Examples below.In particular, enhanced luminescence may be determined by measuring theluminescence signal (RLU) resulting from the incubation of bacterial ormammalian lysates containing CBR variants in question with theluciferase substrate, e.g., a novel luciferin derivative such asPBI-4813 or PBI-4739. The luminescence signal may be compared to that ofa reference point such as CBR with D-luciferin or a luciferin derivativesuch as PBI-4813 or PBI-4739, or Luc2 (firefly) luciferase withD-luciferin.

In certain embodiments, the CBR variant has increased luminescenceemission, e.g., at least about 1-fold, at least about 2-fold, at leastabout 3-fold, at least about 4-fold, at least about 5-fold, at leastabout 6-fold, at least about 7-fold, at least about 8-fold, at leastabout 9-fold, or at least about 10-fold in a prokaryotic cell and/or aeukaryotic cell relative to the corresponding reference luciferase, suchas a CBR of SEQ ID NO: 1, using the same or different substrate, such asD-luciferin, PBI-4813 or PBI-4739. In some embodiments, one or moreproperties of the CBR variant are compared to comparable properties of aluciferase from another species, e.g. a firefly luciferase or a Renillaluciferase.

b. Altered Light Emission Spectra

The CBR variant polypeptide may have altered light emission spectracompared to a parental CBR, e.g., SEQ ID NO: 1. The CBR variantpolypeptide may be able to emit light at a longer wavelength when aluciferin is utilized by the CBR variant polypeptide to generateluminescence. The CBR variant polypeptide is able to emit light at alonger wavelength when a luciferin derivative, such as PBI-4813 orPBI-4739, is utilized by the CBR variant polypeptide to generateluminescence.

The CBR variant polypeptide may emit light having a shift in spectralmaximum relative to the light produced by the CBR polypeptide of SEQ IDNO: 1 using a luciferin or novel luciferin derivative such as PBI-4813or PBI-4739. In some embodiments, the CBR variant polypeptide may emitlight having an increased or decreased shift in spectral maximum of atleast about 1 nm to at least about 200 nm, at least about 2 nm to atleast about 200 nm, at least about 3 nm to at least about 200 nm, atleast about 4 nm to at least about 200 nm, at least about 5 nm to atleast about 200 nm, at least about 6 nm to at least about 200 nm, atleast about 7 nm to at least about 200 nm, at least about 8 nm to atleast about 200 nm, at least about 9 nm to at least about 200 nm, atleast about 10 nm to at least about 200 nm, at least about 15 nm to atleast about 200 nm, at least about 20 nm to at least about 200 nm, atleast about 30 nm to at least about 200 nm, at least about 40 nm to atleast about 200 nm, at least about 50 nm to at least about 200 nm, atleast about 60 nm to at least about 200 nm, at least about 70 nm to atleast about 200 nm, at least about 80 nm to at least about 200 nm, atleast about 90 nm to at least about 200 nm, at least about 100 nm to atleast about 200 nm, at least about 150 nm to at least about 200 nm, atleast about 1 nm to at least about 150 nm, at least about 2 nm to atleast about 150 nm, at least about 3 nm to at least about 150 nm, atleast about 4 nm to at least about 150 nm, at least about 5 nm to atleast about 150 nm, at least about 6 nm to at least about 150 nm, atleast about 7 nm to at least about 150 nm, at least about 8 nm to atleast about 150 nm, at least about 9 nm to at least about 150 nm, atleast about 10 nm to at least about 150 nm, at least about 15 nm to atleast about 150 nm, at least about 20 nm to at least about 150 nm, atleast about 30 nm to at least about 150 nm, at least about 40 nm to atleast about 150 nm, at least about 50 nm to at least about 150 nm, atleast about 60 nm to at least about 150 nm, at least about 70 nm to atleast about 150 nm, at least about 80 nm to at least about 150 nm, atleast about 90 nm to at least about 150 nm, at least about 100 nm to atleast about 150 nm, at least about 1 nm to at least about 100 nm, atleast about 2 nm to at least about 100 nm, at least about 3 nm to atleast about 100 nm, at least about 4 nm to at least about 100 nm, atleast about 5 nm to at least about 100 nm, at least about 6 nm to atleast about 100 nm, at least about 7 nm to at least about 100 nm, atleast about 8 nm to at least about 100 nm, at least about 9 nm to atleast about 100 nm, at least about 10 nm to at least about 100 nm, atleast about 15 nm to at least about 100 nm, at least about 20 nm to atleast about 100 nm, at least about 30 nm to at least about 100 nm, atleast about 40 nm to at least about 100 nm, at least about 50 nm to atleast about 100 nm, at least about 60 nm to at least about 100 nm, atleast about 70 nm to at least about 100 nm, at least about 80 nm to atleast about 100 nm, at least about 90 nm to at least about 100 nm, atleast about 1 nm to at least about 70 nm, at least about 2 nm to atleast about 70 nm, at least about 3 nm to at least about 70 nm, at leastabout 4 nm to at least about 70 nm, at least about 5 nm to at leastabout 70 nm, at least about 6 nm to at least about 70 nm, at least about7 nm to at least about 70 nm, at least about 8 nm to at least about 70nm, at least about 9 nm to at least about 70 nm, at least about 10 nm toat least about 70 nm, at least about 15 nm to at least about 70 nm, atleast about 20 nm to at least about 70 nm, at least about 30 nm to atleast about 70 nm, at least about 40 nm to at least about 70 nm, atleast about 50 nm to at least about 70 nm, at least about 60 nm to atleast about 70 nm, at least about 1 nm to at least about 50 nm, at leastabout 2 nm to at least about 50 nm, at least about 3 nm to at leastabout 50 nm, at least about 4 nm to at least about 50 nm, at least about5 nm to at least about 50 nm, at least about 6 nm to at least about 50nm, at least about 7 nm to at least about 50 nm, at least about 8 nm toat least about 50 nm, at least about 9 nm to at least about 50 nm, atleast about 10 nm to at least about 50 nm, at least about 15 nm to atleast about 50 nm, at least about 20 nm to at least about 50 nm, atleast about 30 nm to at least about 50 nm, at least about 40 nm to atleast about 50 nm, at least about 1 nm to at least about 20 nm, at leastabout 2 nm to at least about 20 nm, at least about 3 nm to at leastabout 20 nm, at least about 4 nm to at least about 20 nm, at least about5 nm to at least about 20 nm, at least about 6 nm to at least about 20nm, at least about 7 nm to at least about 20 nm, at least about 8 nm toat least about 20 nm, at least about 9 nm to at least about 20 nm, atleast about 10 nm to at least about 20 nm, or at least about 15 nm to atleast about 20 nm, relative to the light produced by the CBR polypeptideof SEQ ID NO: 1. The CBR variant polypeptide may emit light having anincreased or decreased shift in spectral maximum of at least about 1 nm,at least about 2 nm, at least about 3 nm, at least about 4 nm, at leastabout 5 nm, at least about 6 nm, at least about 7 nm, at least about 8nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, atleast about 20 nm, at least about 30 nm, at least about 40 nm, at leastabout 50 nm, at least about 60 nm, at least about 70 nm, at least about80 nm, at least about 90 nm, at least about 100 nm, at least about 150nm, or at least about 200 nm relative to the light produced by the CBRpolypeptide of SEQ ID NO: 1.

The CBR variant polypeptide may emit light having a spectral maximumbetween about 650 nm to about 1000 nm, between about 700 nm to about1000 nm, between about 725 nm to about 1000 nm, between about 750 nm toabout 1000 nm, between about 775 nm to about 1000 nm, between about 800nm to about 1000 nm, between about 850 nm to about 1000 nm, betweenabout 900 nm to about 1000 nm, between about 950 nm to about 1000 nm,about 650 nm to about 900 nm, between about 700 nm to about 900 nm,between about 725 nm to about 900 nm, between about 750 nm to about 900nm, between about 775 nm to about 900 nm, between about 800 nm to about900 nm, between about 850 nm to about 900 nm, about 650 nm to about 800nm, between about 700 nm to about 800 nm, between about 725 nm to about800 nm, between about 750 nm to about 800 nm, between about 775 nm toabout 800 nm, about 650 nm to about 775 nm, between about 700 nm toabout 775 nm, between about 725 nm to about 775 nm, between about 750 nmto about 775 nm, between about 650 nm to about 750 nm, between about 700nm to about 750 nm, between about 725 nm to about 750 nm, between about650 nm to about 725 nm, between about 700 nm to about 725 nm, or betweenabout 725 nm to about 750 nm when a luciferin or novel luciferinderivative, such as PBI-4813 or PBI-4739, is used as a substrate. TheCBR variant polypeptide may emit light having a spectral maximum of atleast about 600 nm, at least about 650 nm, at least about 700 nm, atleast about 725 nm, at least about 750 nm, at least about 775 nm, atleast about 800 nm, at least about 900 nm, or at least about 1000 nmwhen a luciferin or novel luciferin derivative, such as PBI-4813 orPBI-4739, is used as a substrate.

c. Altered Substrate Specificity

The CBR variant polypeptide may have altered substrate specificitycompared to a CBR polypeptide of SEQ ID NO: 1. The CBR variantpolypeptide may have increased or decreased substrate specificity for aluciferin or novel luciferin derivative such as PBI-4739 or PBI-4813,compared to a CBR polypeptide of SEQ ID NO: 1. The CBR variantpolypeptide may have a change, such as an increase or decrease, inrelative specificity relative to the CBR variant polypeptide in thepresence of a luciferin compared to a luciferin derivative, such asPBI-4739 or PBI-4813. The CBR variant polypeptide may have a change,such as an increase or decrease, in relative specificity relative to theCBR variant polypeptide in the presence of a luciferin derivativecompared to a different luciferin derivative, such as PBI-4739 orPBI-4813.

The CBR variant polypeptide may have an altered Km or Vmax compared to aCBR polypeptide of SEQ ID NO: 1 using D-luciferin and/or a novelluciferin derivative. A CBR variant polypeptide having a lower Km for aparticular substrate may be an advantage in in vivo imaging where it isdifficult to “saturate” the substrate.

The CBR variant may have a Km for PBI-4813 of at least about 0.10 μM, atleast about 0.20 μM, at least about 0.30 μM, at least about 0.40 μM, atleast about 0.50 μM, at least about 0.60 μM, at least about 0.70 μM, atleast about 0.80 μM, at least about 0.81 μM, at least about 0.82 μM, atleast about 0.83 μM, at least about 0.84 μM, at least about 0.85 μM, atleast about 0.86 μM, at least about 0.87 μM, at least about 0.88 μM, atleast about 0.89 μM, at least about 0.90 μM, at least about 1.00 μM, atleast about 1.50 μM, at least about 2.00 μM, at least about 2.10 μM, atleast about 2.20 μM, at least about 2.30 μM, at least about 2.40 μM, atleast about 2.50 μM, at least about 2.60 μM, at least about 2.70 μM, atleast about 2.80 μM, at least about 2.90 μM, at least about 3.00 μM, atleast about 4.00 μM, or at least about 5.00 μM, or has a Km ranging fromat least about 0.01 μM to at least about 5.00 μM, at least about 0.01 μMto at least about 4.00 μM, at least about 0.01 μM to at least about 3.00μM, at least about 0.01 μM to at least about 2.50 μM, at least about0.01 μM to at least about 2.00 μM, at least about 0.01 μM to at leastabout 1.00 μM, at least about 0.01 μM to at least about 0.80 μM, atleast about 0.01 μM to at least about 0.50 μM, at least about 0.05 μM toat least about 5.00 μM, at least about 0.05 μM to at least about 4.00μM, at least about 0.05 μM to at least about 3.00 μM, at least about0.05 μM to at least about 2.50 μM, at least about 0.05 μM to at leastabout 2.00 μM, at least about 0.05 μM to at least about 1.00 μM, atleast about 0.05 μM to at least about 0.80 μM, at least about 0.08 μM toat least about 5.00 μM, at least about 0.08 μM to at least about 4.00μM, at least about 0.08 μM to at least about 3.00 μM, at least about0.08 μM to at least about 2.50 μM, at least about 0.08 μM to at leastabout 2.00 μM, or at least about 0.08 μM to at least about 1.00 μM.

The CBR variant may have a Km for PBI-4739 of at least about or has a Kmranging from at least about 0.10 μM, at least about 0.50 μM, at leastabout 1.00 μM, at least about 1.50 μM, at least about 2.00 μM, at leastabout 2.10 μM, at least about 2.20 μM, at least about 2.30 μM, at leastabout 2.40 μM, at least about 2.50 μM, at least about 2.60 μM, at leastabout 2.70 μM, at least about 2.80 μM, at least about 2.90 μM, at leastabout 3.00 μM, at least about 3.10 μM, at least about 3.20 μM, at leastabout 3.30 μM, at least about 3.40 μM, at least about 3.50 μM, at leastabout 3.60 μM, at least about 3.70 μM, at least about 3.80 μM, at leastabout 3.90 μM, at least about 4.00 μM, or at least about 5.00 μM, or hasa Km ranging from at least about 0.01 μM to at least about 5.00 μM, atleast about 0.01 μM to at least about 4.00 μM, at least about 0.01 μM toat least about 3.00 μM, at least about 0.01 μM to at least about 2.50μM, at least about 0.01 μM to at least about 2.00 μM, at least about0.01 μM to at least about 1.00 μM, at least about 0.01 μM to at leastabout 0.80 μM, at least about 0.01 μM to at least about 0.50 μM, 1.00 μMto at least about 5.00 μM, at least about 1.00 μM to at least about 4.00μM, at least about 1.00 μM to at least about 3.00 μM, at least about1.00 μM to at least about 2.50 μM, at least about 1.00 μM to at leastabout 2.00 μM, 2.00 μM to at least about 5.00 μM, at least about 2.00 μMto at least about 4.00 μM, at least about 2.00 μM to at least about 3.00μM, at least about 2.00 μM to at least about 2.50 μM, 3.00 μM to atleast about 5.00 μM, or at least about 3.00 μM to at least about 4.00μM.

The CBR variant may have a Km for PBI-4813 that is at least about1-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 15-fold, at least about 20-fold, at least about30-fold, at least about 4-fold, at least about 50-fold, at least about60-fold, at least about 70-fold, at least about 80-fold, at least about90-fold, at least about 100-fold lower, at least about 150-fold, atleast about 200-fold, at least about 300-fold, at least about 400-fold,at least about 500-fold, or at least about 1000-fold lower than the Kmof a CBR polypeptide of SEQ ID NO: 1 for PBI-4813.

The CBR variant may have a Km for PBI-4813 that is at least about1-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 15-fold, at least about 20-fold, at least about30-fold, at least about 4-fold, at least about 50-fold, at least about60-fold, at least about 70-fold, at least about 80-fold, at least about90-fold, at least about 100-fold lower, at least about 150-fold, atleast about 200-fold, at least about 300-fold, at least about 400-fold,at least about 500-fold, or at least about 1000-fold lower than the Kmof a CBR polypeptide of SEQ ID NO: 1 for D-luciferin.

The CBR variant may have a Km for PBI-4739 that is at least about1-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 15-fold, at least about 20-fold, at least about30-fold, at least about 4-fold, at least about 50-fold, at least about60-fold, at least about 70-fold, at least about 80-fold, at least about90-fold, at least about 100-fold lower, at least about 150-fold, atleast about 200-fold, at least about 300-fold, at least about 400-fold,at least about 500-fold, or at least about 1000-fold lower than the Kmof a CBR polypeptide of SEQ ID NO: 1 for PBI-4739.

The CBR variant may have a Km for PBI-4739 that is at least about1-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 15-fold, at least about 20-fold, at least about30-fold, at least about 4-fold, at least about 50-fold, at least about60-fold, at least about 70-fold, at least about 80-fold, at least about90-fold, at least about 100-fold lower, at least about 150-fold, atleast about 200-fold, at least about 300-fold, at least about 400-fold,at least about 500-fold, or at least about 1000-fold lower than the Kmof a CBR polypeptide of SEQ ID NO: 1 for D-luciferin.

The CBR variant may have a relative Vmax for PBI-4813 that is at leastabout 1-fold, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 6-fold, at leastabout 7-fold, at least about 8-fold, at least about 9-fold, at leastabout 10-fold, at least about 15-fold, at least about 20-fold, at leastabout 30-fold, at least about 4-fold, at least about 50-fold, at leastabout 60-fold, at least about 70-fold, at least about 80-fold, at leastabout 90-fold, at least about 100-fold lower, at least about 150-fold,at least about 200-fold, at least about 300-fold, at least about400-fold, at least about 500-fold, or at least about 1000-fold higherthan the relative Vmax of a CBR polypeptide of SEQ ID NO: 1 forPBI-4813.

The CBR variant may have a relative Vmax for PBI-4739 that is at leastabout 1-fold, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 6-fold, at leastabout 7-fold, at least about 8-fold, at least about 9-fold, at leastabout 10-fold, at least about 15-fold, at least about 20-fold, at leastabout 30-fold, at least about 4-fold, at least about 50-fold, at leastabout 60-fold, at least about 70-fold, at least about 80-fold, at leastabout 90-fold, at least about 100-fold lower, at least about 150-fold,at least about 200-fold, at least about 300-fold, at least about400-fold, at least about 500-fold, or at least about 1000-fold higherthan the relative Vmax of a CBR polypeptide of SEQ ID NO: 1 forPBI-4739.

d. Fusion Proteins

In some embodiments, the CBR variant of the invention has one or moreheterologous amino acid sequences at the N-terminus, C-terminus, or both(a fusion polypeptide such as one with an epitope or fusion tag), whichoptionally directly or indirectly interact with a molecule of interest.In some embodiments, the presence of the heterologous sequence(s) doesnot substantially alter the luminescence of the CBR variant eitherbefore or after the interaction with the molecule of interest. Theheterologous amino acid sequence may be any protein of interest such asRNasin or RNase, and/or a channel protein, a receptor, a membraneprotein, a cytosolic protein, a nuclear protein, a structural protein, aphosphoprotein, a kinase, a signaling protein, a metabolic protein, amitochondrial protein, a receptor associated protein, a fluorescentprotein, an enzyme substrate, a transcription factor, a transporterprotein and/or a targeting sequence, e.g., a myristilation sequence, amitochondrial localization sequence, or a nuclear localization sequence,that directs the hydrolase fragment, for example, a fusion protein, to aparticular location.

In some embodiments, the heterologous amino acid sequence is an epitopetag. In some embodiments, the heterologous amino acid sequence is onewhich, during or after interaction with a molecule of interest,undergoes a conformational change, which in turn alters the activity ofthe CBR variant e.g., a CBR variant with such an amino acid sequence isuseful to detect allosteric interactions. The CBR variant or a fusionwith the CBR variant or a fragment thereof may be employed as areporter.

The CBR variants of the present invention may be coupled to any proteinof interest or molecule of interest. In some embodiments, the variantsare fusion proteins, for example some variants are coupled to a HALOTAG®polypeptide (also referred to as “HT7”) attached at either theN-terminus or the C-terminus. In some embodiments, a fusion or chimericprotein contains a CBR variant joined at the N-terminus to a HALOTAG®fusion protein (Promega). In other embodiments, a fusion or chimericprotein contains a CBR variant joined at the C-terminus to a HALOTAG®fusion protein. Signal sequences, in combination with membrane anchoringsequences, may be used to position or display CBR variants on the outersurface of the cellular membrane. Other methods, known in the art mayalso be used to position CBR variants to the membrane or other locationswithin the cell.

e. Vectors and Host Cells Encoding the Modified Luciferase or FusionsThereof

Once a desirable nucleic acid molecule encoding a CBR variant or afragment thereof, such as one with luminescence activity or which may becomplemented by another molecule to result in luminescence activity, ora fusion thereof with luminescence activity, is prepared, an expressioncassette encoding the CBR variant or a fragment thereof, e.g., one forcomplementation, or a fusion thereof with luminescence activity, may beprepared. For example, a nucleic acid molecule comprising a nucleic acidsequence encoding a CBR variant is optionally operably linked totranscription regulatory sequences, e.g., one or more enhancers, apromoter, a transcription termination sequence or a combination thereof,to form an expression cassette. The nucleic acid molecule or expressioncassette may be introduced to a vector, e.g., a plasmid or viral vector,which optionally includes a selectable marker gene, and the vectorintroduced to a cell of interest, for example, a prokaryotic cell suchas E. coli, Streptomyces spp., Bacillus spp., Staphylococcus spp. andthe like, as well as eukaryotic cells including a plant (dicot ormonocot), fungus (including yeast, e.g., Pichia, Saccharomyces orSchizosaccharomyces), or a mammalian cell, lysates thereof, or to an invitro transcription/translation mixture. Mammalian cells include but arenot limited to bovine, caprine, ovine, canine, feline, non-humanprimate, e.g., simian, and human cells. Mammalian cell lines include,but are not limited to, CHO, COS, HEK293, HeLa, CV-1, SH-SY5Y, and NIH3T3 cells, although numerous other cell lines can also be used as well.

The expression of an encoded CBR variant may be controlled by anypromoter capable of expression in prokaryotic cells or eukaryotic cellsincluding synthetic promoters. Prokaryotic promoters include, but arenot limited to, SP6, T7, T5, tac, bla, trp, gal, lac or maltosepromoters, including any fragment that has promoter activity. Eukaryoticpromoters include, but are not limited to, constitutive promoters, e.g.,viral promoters such as CMV, SV40 and RSV promoters, as well asregulatable promoters, e.g., an inducible or repressible promoter suchas the Tet promoter, the hsp70 promoter and a synthetic promoterregulated by CRE, including any fragment that has promoter activity. Theexpression of an encoded CBR variant may also be controlled bypost-transcriptional processes, such as by regulation of RNA processingor regulation of translation, for example by RNAi, miRNA, shRNA, siRNA,or by RNA or protein degradation. The nucleic acid molecule, expressioncassette and/or vector of the invention may be introduced to a cell byany method including, but not limited to, calcium-mediatedtransformation, electroporation, microinjection, lipofection, and thelike.

f. Optimized Sequences Encoding the CBR Variants

Also provided is an isolated nucleic acid molecule (polynucleotide)comprising a nucleic acid sequence encoding a CBR variant of theinvention, a functional fragment thereof or a fusion protein thereof. Insome embodiments, the isolated nucleic acid molecule comprises a nucleicacid sequence which is optimized for expression in at least one selectedhost. Optimized sequences include sequences which are codon optimized,i.e., codons which are employed more frequently in one organism relativeto another organism, e.g., a distantly related organism, as well asmodifications to add or modify Kozak sequences and/or introns, and/or toremove undesirable sequences, for instance, potential transcriptionfactor binding sites. Such optimized sequences can provide enhancedexpression, e.g. increased levels of protein expression, when introducedinto a host cell. Examples of optimized sequences are disclosed in U.S.Pat. No. 7,728,118 and U.S. Pat. Appl. Publ. Nos. 2008/0070299 and2008/0090291, each of which is incorporated by reference herein.

In some embodiments, the polynucleotide includes a nucleic acid sequenceencoding a CBR variant of the invention, which nucleic acid sequence isoptimized for expression in a mammalian host cell in culture (e.g., CHOcells), in a living animal (e.g., mouse), or in a tissue-specific cellin a living animal. In some embodiments, the polynucleotide may includea codon optimized sequence of any one of SEQ ID NOs: 2-4. In someembodiments, the polynucleotide may include a polynucleotide sequence ofany one of SEQ ID NOs: 7-9. In some embodiments, the polynucleotide mayinclude a polynucleotide sequence of SEQ ID NO: 10. In some embodiments,the nucleic acid sequence is optimized for expression in a bacterialcell or plant. In some embodiments, an optimized polynucleotide nolonger hybridizes to the corresponding non-optimized sequence, e.g.,does not hybridize to the non-optimized sequence under medium or highstringency conditions. The term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “medium” or “low”stringency are often used when it is desired that nucleic acids that arenot completely complementary to one another be hybridized or annealedtogether. The art knows well that numerous equivalent conditions can beemployed to comprise medium or low stringency conditions.

In some embodiments, the polynucleotide has less than 90%, e.g., lessthan 80%, nucleic acid sequence identity to the correspondingnon-optimized sequence and optionally encodes a polypeptide having atleast 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100%, amino acid sequence identity with the polypeptideencoded by the non-optimized sequence. Constructs, e.g., expressioncassettes, and vectors comprising the isolated nucleic acid molecule,e.g., with optimized nucleic acid sequence, as well as kits comprisingthe isolated nucleic acid molecule, construct or vector are alsoprovided.

A nucleic acid molecule comprising a nucleic acid sequence encoding aCBR variant of the invention, a fragment thereof or a fusion thereof isoptionally optimized for expression in a particular host cell and alsooptionally operably linked to transcription regulatory sequences, e.g.,one or more enhancers, a promoter, a transcription termination sequenceor a combination thereof, to form an expression cassette.

In some embodiments, a nucleic acid sequence encoding a CBR variant ofthe invention, a fragment thereof or a fusion thereof is optimized byreplacing codons, e.g., at least 25% of the codons in a parental CBRsequence with codons which are preferentially employed in a particular(selected) cell. Preferred codons have a relatively high codon usagefrequency in a selected cell, and preferably their introduction resultsin the introduction of relatively few transcription factor binding sitesfor transcription factors present in the selected host cell, andrelatively few other undesirable structural attributes. Examples ofundesirable structural attributes include, but not limited to,restriction enzyme sites, eukaryotic sequence elements, vertebratepromoter modules and transcription factor binding sites, responseelements, E. coli sequence elements, mRNA secondary structure. Thus, theoptimized nucleic acid product may have an improved level of expressiondue to improved codon usage frequency, and a reduced risk ofinappropriate transcriptional behavior due to a reduced number ofundesirable transcription regulatory sequences.

An isolated and optimized nucleic acid molecule may have a codoncomposition that differs from that of the corresponding wild-typenucleic acid sequence at more than 30%, 35%, 40% or more than 45%, e.g.,50%, 55%, 60% or more of the codons. Exemplary codons for use in theinvention are those which are employed more frequently than at least oneother codon for the same amino acid in a particular organism and, insome embodiments, are also not low-usage codons in that organism and arenot low-usage codons in the organism used to clone or screen for theexpression of the nucleic acid molecule. Moreover, codons for certainamino acids (i.e., those amino acids that have three or more codons),may include two or more codons that are employed more frequently thanthe other (non-preferred) codon(s). The presence of codons in thenucleic acid molecule that are employed more frequently in one organismthan in another organism results in a nucleic acid molecule which, whenintroduced into the cells of the organism that employs those codons morefrequently, is expressed in those cells at a level that is greater thanthe expression of the wild-type or parent nucleic acid sequence in thosecells.

In some embodiments of the invention, the codons that are different arethose employed more frequently in a mammal, while in still otherembodiments, the codons that are different are those employed morefrequently in a plant or bacteria. Preferred codons for differentorganisms are known to the art, e.g., seehttp://www.kazusa.or.jp./codon/. A particular type of mammal, e.g., ahuman, may have a different set of preferred codons than another type ofmammal. Likewise, a particular type of plant or bacteria may have adifferent set of preferred codons than another type of plant orbacteria. In some embodiments of the invention, the majority of thecodons that differ are ones that are preferred codons in a desired hostcell. Preferred codons for organisms including mammals (e.g., humans)and plants are known to the art (e.g., Wada et al., Nucl. Acids Res.,18:2367 (1990); Murray et al., Nucl. Acids Res., 17:477 (1989)).

3. NEAR-INFRARED BIOLUMINESCENCE SYSTEMS

The present disclosure also provides near-IR bioluminescence systemsthat include the CBR variants, described above, and a novel luciferinderivative. Certain combinations of the CBR variants and novel luciferinderivatives provide significant technical advantages for bioluminescentassays especially for in depth tissue imaging because near-IRluminescence is generated. The disclosed invention provides enhanceddetection of signals from deep animal tissues, in part, because of thelonger wavelength are not absorbed by the tissues, but also becausethere is an improvement in light emission over the parental CBR andknown luciferin substrates. In addition, other reagents may be includedto bioluminescence systems, including but not limited to those thatinhibit or prevent inactivation of the CBR variant or otherwise extendor enhance luminescent signal.

a. Novel Luciferin Derivatives

The near-IR bioluminescence systems may include novel red-shiftedluciferin derivatives, such as those disclosed in PCT application No.PCT/US2014/021678, which is incorporated herein by reference in itsentirety. For example, the luciferin derivative may include a compoundof one of Formulas (Ia), (Ib), (Ic), (II), (III), (IV), (V), (VI), or(VII), or pro-substrates thereof, such as Reductase Substrates,Glycosidase Substrates, Protease and Protease-Dependent ProteinModifying Substrates, Oxidase Substrates, Carboxyl-Based Pro-Substrates,Glutathione Transferase Substrate, Beta-Lactamase Substrates, and OtherPro-Substrates. In some embodiments, the novel luciferin derivative maybe PBI-4739 or PBI-4813 (see FIG. 1; disclosed in PCT Application No.PCT/US2014/021678).

(1) Compound of Formula(s) (Ia), (Ib), (Ic), (II), (III), (IV), (V),(VI), or (VII)

The luciferin derivative may include a compound according to Formulas(Ia), (Ib) and (Ic):

whereinX is CN or

each Y is independently halo, SO₃H, C₁₋₄ alkyl, substituted C₁₋₄ alkyl,OR¹ or NR¹R²;each R¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl;each R² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a 4 to 8 membered ring;n is 1 to 6;two Y substituents may join together to form a ring containing from 5 to7 ring atoms; andwherein at least one Y is either OH or NR¹R².

The luciferin derivative may include a compound, but are not limited to:

In other embodiments, the luciferin derivative may include a compoundaccording to Formula (II):

whereinX is CN or

Y is OR¹ or NR¹R²;R¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; andR² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a ring.

In a further embodiments, the luciferin derivative may include acompound according to Formula (III):

whereinX is CN or

Y is OR¹ or NR′R²;R¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; andR² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a ring.

In yet other embodiments, the luciferin derivative may include acompound according to Formula (IV):

whereinX is CN or

Y is OR¹ or NR¹R²;R¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; andR² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a ring.

In other embodiments, the luciferin derivative may include a compoundaccording to Formula (V):

whereinX is CN or

Y is OR¹ or NR¹R²;R¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; andR² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a ring.

In other embodiments, the luciferin derivative may include a compoundaccording to Formula (VI):

whereinX is CN or

Y is OR¹ or NR¹R²;R¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; andR² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a ring.

In an additional embodiments, the luciferin derivative may include acompound according to Formula (VII):

whereinX is CN or

each Y is independently OR¹ or NR¹R²;each R¹ is independently H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; andeach R² is independently H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a ring.

In certain embodiments, the emission maximum of the compounds is atleast about 650 nm, about 655 nm, about 680, or about 760 nm.

(2) Pro-Substrates

The present invention also provides compounds which are substrates forvarious non-luciferase enzymes and are pro-substrates for luciferaseenzymes. The non-luciferase enzymes include, but are not limited to,reductases, glycosidases, proteases, peptidases, oxidases, esterases,cytochrome P450s, beta-lactamases, glycosylases and glutathionetransferases.

In some embodiments, these pro-substrates have a substituent that is asubstrate for a non-luciferase position at the one or more Y positionswhich is cleaved to form

whereinX is CN or

each Y is OH or NH₂; andn is 1 to 3.

(a) Reductase Substrates

In some embodiments, the compound is a reductase substrate. In someembodiments, a reductase substrate has the formula:

whereinX is CN or

Y is OR;R is

W is S, NR_(N), or O;Z is S, NR_(N), O or CH; andR_(N) is H, C₁₋₄ alkyl, or substituted C₁₋₄ alkyl.

In some embodiments, a reductase substrate has the formula:

whereinX is CN or

Y is OR¹ or NR¹R²; andR¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl;R² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a 4 to 8 membered ring.

In some embodiments, a reductase substrate has the formula:

whereinX is CN or

Y is OR; andR is

Reductase substrates include, but are not limited to, the followingcompounds:

(b) Glycosidase Substrates

In some embodiments, the compound is a glycosidase substrate. In someembodiments, the glycosidase substrate is a compound of formula:

whereinX is CN or

Y is OR;R is

A is OR or NHAc;each R₅ is independently H, a monosaccharide or a polyethylene glycolmoiety of up to 40 units.

Glycosidase substrates include, but are not limited to, the followingcompounds:

(c) Protease and Protease-Dependent Protein Modifying Substrates

In some embodiments, the compound is a protease or protease-dependentprotein modifying substrate. In some embodiments, the substrate is acompound of formula:

whereinX is CN or

Y is NHR;R is

R₇ is an amino acid side chain;R₆ is H, a nitrogen protecting group, or a chain of up to 20 aminoacids.

Suitable nitrogen protecting groups include, but are not limited to,those traditionally known to those skilled in the art, such as Boc, Cbz,Ac and Fmoc.

These substrates include, but are not limited to, the followingcompounds:

(d) Oxidase Substrates

In some embodiments, the compound is an oxidase substrate. In someembodiments, an oxidase substrate is a compound of formula:

whereinX is —CH(OR₁₀)₂;R₁₀ is C₁₋₄ alkyl, substituted C₁₋₄ alkyl, benzyl, or substitutedbenzyl;Y is OR¹ or NR¹R²; andR¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl;

-   -   R² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; or    -   R¹ and R² together form a 4 to 8 membered ring.

In some embodiments, an oxidase substrate is a compound of formula:

whereinX is CN or

Y is H or OR; andR is C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl, aryl, substituted aryl,aralkyl or substituted aralkyl.

In some embodiments, an oxidase substrate is a compound of formula:

whereinX is —CH(OR₁₀)₂;R₁₀ is C₁₋₄ alkyl, substituted C₁₋₄ alkyl, benzyl or substituted benzyl;Y is OR¹ or NR¹R²; andR¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl;R² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a 4 to 8 membered ring.

Oxidase substrates include, but are not limited to, the followingcompounds:

(e) Carboxyl-Based Pro-Substrates

In some embodiments, the compound is a carboxyl-based pro-substrate. Insome embodiments, the carboxyl-based pro-substrate is a compound offormula:

whereinR₈ is CH₂OH, C(O)R₁₀ or —C(O)ZR₉;Z is O or NH;R₉ is C₁₋₇ alkyl or substituted C₁₋₇ alkyl;R₁₀ is a peptide;Y is OR¹ or NR¹R²; andR¹ is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl;R² is H, C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl; orR¹ and R² together form a 4 to 8 membered ring.

Carboxyl-based pro-substrates include, but are not limited to, thefollowing compounds:

(f) Glutathione Transferase Substrate

In some embodiments, the compound may be a glutathione transferasesubstrate. In some embodiments, the glutathione transferase substrate isa compound of formula:

whereinX is CN or

Y is OR;R is

-   -   each R₁₁ is independently H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl,        CF₃, halogen, NO₂, CO₂R₁₂ or any two adjacent R₁₁ can form a        fused ring provided that at least one of R₁₁ is NO₂; and    -   R₁₂ is H, C₁₋₆ alkyl or substituted C₁₋₆ alkyl.

Glutathione transferase substrates include, but are not limited to, thefollowing compounds:

(g) Beta-Lactamase Substrates

In some embodiments, the compound is a beta-lactamase substrate. In someembodiments, the beta-lactamase substrate is a compound of formula:

whereinX is CN or

Y is O, NH, N(C₁₋₇ alkyl), or N(substituted C₁₋₇ alkyl);Z is absent or O;A is C₁₋₄ alkylene or substituted C₁₋₄ alkylene; andR₁₄ is H, phenacetyl, or a cephalosporin side chain.

Suitable cephalosporin side chains include those known to one ofordinary skill in the art.

Beta-lactamase substrates include, but are not limited to, the followingcompounds:

(h) Other Pro-Substrates

The present invention also provides compounds which react with variousbiologically important small molecules, such as hydrogen peroxide, andare pro-substrates for luciferase enzymes. In some embodiments, thesecompounds are reactive to hydrogen peroxide. In some embodiments, thesecompounds have the formula:

whereinX is CN or

Y is L-R;L is a linker; andR is a boronic acid or borate ester.

In some embodiments, R is —B(OR₁₅)₂; wherein each R₁₅ is independentlyselected from H and C₁₋₄ alkyl. In some embodiments, R is

wherein each R₁₆ and R₁₇ is independently selected from H, C₁₋₄ alkyl,substituted C₁₋₄ alkyl, CF₃, phenyl or substituted phenyl; or R₁₆ andR₁₇ together can be an alkyl ring having from 3-7 carbons or can bereplaced by a fused 6-membered aromatic ring.

In some embodiments, the linker is a direct bond. In other embodiments,the linker is

wherein

A is —C₆(R₂₀)₄—, —O—C₆(R₂₀)₄— or —(CR₂₁═CR₂₁)_(n)— or —S—C₆(R₂₀)₄— or—NR′—C₆(R₂₀)₄ or a direct bond;

R′ is H, C₁₋₄ alkyl, or substituted C₁₋₄ alkyl;

each R₂₃ is independently halo, H, C₁₋₄ alkyl, substituted C₁₋₄ alkyl,C₁₋₄ hydroxyalkyl, substituted C₁₋₄ hydroxyalkyl, C₁₋₄ alkylcarboxylicacid or substituted C₁₋₄ alkylcarboxylic acid;

each R₂₀ is independently H, halo, CH₃, OCH₃, or NO₂;

each R₂₁ is independently H or CH₃;

n is 1 or 2; and

X is a selected from —O—,

These compounds include, but are not limited to, the followingcompounds:

4. METHODS OF USING THE CBR VARIANTS AND/OR BIOLUMINESCENT SYSTEMS

The CBR variants and near-IR bioluminescence systems may be used in anyway that luciferases and luciferase substrates, e.g., luciferin andluciferin derivatives, have been used. The CBR variants and/or near-IRbioluminescent systems may be used with a sample (including cells,tissues, animals, etc.) or with in vivo imaging and detected usingvarious microscopy and imaging techniques. The CBR variants and/ornear-IR bioluminescent systems may be used as a transcriptional reporteror as a biosensor. The CBR variants and/or near-IR bioluminescentsystems may be used in dual-color assays or multiplexing. The CBRvariants and/or near-IR bioluminescent systems may be used in assays todetect the presence or activity of enzymes such as non-luminescentenzymes. For example, they may be used in a bioluminogenic method whichemploys an analog of luciferins to detect one or more molecules in asample, e.g., an enzyme, a cofactor for an enzymatic reaction, an enzymesubstrate, an enzyme inhibitor, an enzyme activator, or OH radicals, orone or more conditions, e.g., redox conditions. In some embodiments, theCBR variants and/or near-IR bioluminescent systems may be used as anenergy donor to another molecule (e.g., to a fluorophore, a chromophore,or a nanoparticle). The CBR variants and near-IR bioluminescence systemsmay be used in protein proximity assays or protein complementationassays. The disclosed CBR variants and/or near-IR bioluminescent systemsof the invention are also useful in in situ methods of analyzing cells.Methods of performing in situ analysis of cells using a luciferase areknown in the art. The disclosed CBR variants and/or near-IRbioluminescent systems of the invention may be used to distinguishbetween substrates and inhibitors of an enzyme. The screening may beperformed either in vitro or in vivo.

The sample may include an animal (e.g., a vertebrate), a plant, afungus, physiological fluid (e.g., blood, plasma, urine, mucoussecretions and the like), a cell, a cell lysate, a cell supernatant, ora purified fraction of a cell (e.g., a subcellular fraction). Thepresence, amount, spectral distribution, emission kinetics, or specificactivity of such a molecule may be detected or quantified. The moleculemay be detected or quantified in solution, including multiphasicsolutions (e.g., emulsions or suspensions), or on solid supports (e.g.,particles, capillaries, or assay vessels).

The luciferase reaction may be restricted or limited by time, enzymeconcentration, and/or substrate concentration. Reaction conditions maybe adjusted so that the luciferase reaction is carried out underconditions that result in about, at least about, or at most about 20,30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% completion, orany range derivable therein. For example, the reaction may be carriedout at a temperature between about 20° C. and about 45° C., about 20° C.and about 40° C., about 20° C. and about 35° C., about 20° C. and about30° C., about 20° C. and about 25° C., between about 25° C. and about45° C., about 25° C. and about 40° C., about 25° C. and about 35° C.,about 25° C. and about 30° C., between about 30° C. and about 45° C.,about 30° C. and about 40° C., about 30° C. and about 35° C. C, betweenabout 35° C. and about 45° C., or between about 35° C. and about 40° C.The reaction may be carried out at 20° C., 21° C., 22° C., 23° C., 24°C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33°C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42°C., 43° C., 44° C., or 45° C. These temperature condition and/orreaction may be maintained or measured for 1 min, 5 min, 10 min, 15 min,20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min,65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 100 min, 110 min, 120min, 130 min, 140 min, 150 min, 160 min, 170 min, 180 min, 190 min, 200min, 210 min, 220 min, 230 min, 240 min, 250 min, or more.

a. In vivo Imaging

The CBR variants and near-IR (NIR) bioluminescence systems may be usedfor in vivo imaging and may improve the speed, detection limit, anddepth penetration of bioluminescence imaging. The CBR variants andnear-IR bioluminescence systems provide a means for non-invasive animalimaging for a variety of applications, e.g. understanding tumor biology,evaluation of potential therapeutic compounds. For example, the methodsdescribed herein can be used for the rapid and inexpensive evaluation oftumor progression and response to anti-cancer therapeutics in smallanimals, e.g., using transgenic non-human animals, e.g., mice, thatexpress a luciferase reporter gene linked to a promoter or gene that isexpressed, e.g., selectively expressed, in the cells that are desired tobe imaged. Expression of a selected protein of interest may be imaged inreal time in a living cell or animal, using a cell or transgenic animalthat expresses a reporter construct including a nucleic acid encoding aCBR variant linked in frame to a nucleic acid encoding the selectedprotein of interest, or to the promoter for the selected protein.

The methods may be performed on cells or animals (e.g., non-humanmammals, e.g., experimental animals, such as rodents, e.g., rats ormice) that express a CBR variant reporter construct. Generating suchcells or animals may be performed using standard molecular biologicaltechniques. Sufficient amounts of the novel luciferin derivativedescribed herein may be added or administered to the cells or animals,and images of the NIR bioluminescence may be obtained using standardimaging methods. The promoter activity, protein expression, proteinsubcellular localization, protein translocation, and protein half-lifemay be evaluated in real time in living cells and animals.

When an experimental animal is used, the cells containing the NIRbioluminescence can be identified and excised, and evaluated further,e.g., using assays for gene expression, protein expression, or othergenetic or biochemical parameters. The bioluminescence system may bedesigned with another luciferin/luciferase pair (e.g., with differentemission maxima) to allow for simultaneous imaging of bioluminescencefrom two or more luciferases.

(1) Imaging Methods

The methods described herein can be practiced with any imaging systemthat can detect near infrared bioluminescence. Common imaging systemsare available from Xenogen (e.g., IVIS), Hamamatsu, Roper, and Kodak.

(2) Live Cell

In various embodiments, the CBR variants can be used for detectingluminescence in live cells. In some embodiments, a CBR variant can beexpressed in cells (as a reporter or otherwise), and the cells treatedwith a luciferase substrate, e.g., luciferin, luciferin derivative,e.g., a novel luciferin derivative such as PBI-4739 or PBI-4813, orfunctional analog, which may permeate cells in culture, react with theCBR variant and generate luminescence. PBI-4739 or PBI-4813 showcomparable non-toxicity to D-luciferin in terms of cell toxicitystudies. In some embodiments, versions of PBI-4739 or PBI-4813containing chemical modifications known to increase the stability ofnative luciferin derivative in media may be synthesized and used formore robust, live cell CBR variant-based reporter assays. In still otherembodiments, a sample (including cells, tissues, animals, etc.)containing a CBR variant and/or a novel luciferin derivative of thepresent invention may be assayed using various microscopy and imagingtechniques. In still other embodiments, a secretable CBR variant may beexpressed in cells as part of a live-cell reporter system.

b. Use as Transcriptional Reporters

The CBR variants and near-IR bioluminescence systems may be used asgenetic transcriptional reporter systems. The CBR variant or fragmentthereof may be used to study transcriptional expression patterns of anypromoter and/or gene, such as genes involved in development. In someembodiments, the CBR variant or fragment thereof could be operablylinked to transcription regulatory sequences, e.g., one or moreenhancer, a promoter, a transcription termination sequence or acombination thereof, to form an expression cassette. For example, theCBR variant could be operably linked to a minimal promoter and acAMP-response element (CRE).

In certain embodiments, provided is a method for measuring the activityof a promoter in a sample, wherein the promoter is operably linked to agene encoding a CBR variant enzyme. The method includes (a) contactingthe sample with a luciferin or novel luciferin derivative; and (b)determining the activity of the promoter by measuring luminescence ofthe sample, wherein the sample comprises the promoter. The promoter maybe operably linked to the gene via a translational or transcriptionalfusion. A biological pathway of interest, for example, may be examinedby treating a cell that comprises the promoter, which is operably linkedto a gene encoding the luminescent enzyme, with an inducer agent of thepathway. This promoter activity may then be measured and monitored tostudy any correlation between the activity of the promoter and thepathway of interest, as well as obtain kinetic measurements relating togene expression (e.g. inducibility, repression and activation).

c. Multiplexing

The CBR variant and bioluminescence system may be used in a multiplexedreaction with another enzyme (e.g. a luciferase) that emits light at adifferent wavelength. In some embodiments, the CBR variant may be usedas a transcriptional reporter and paired with a luciferase that emitslight at a different wavelength contained in an assay reagent. In someembodiments, the CBR variant may be used with one or more additionalluciferases, where the luminescence of each luciferase may be separatelymeasured through the use of selective enzyme inhibitors. For example,the luminescence of the CBR variant may be measured upon addition ofappropriate substrates and buffers, followed by measurement of a secondluciferase upon a subsequent addition of appropriate substrates andbuffers and one or more inhibitors selective for the first luciferase.

In some embodiments, the CBR variant of the present invention may beused as a functional reporter for a particular gene and the secondenzyme in the multiplex reaction may be used as a functional reporterfor a second gene. In some embodiments, the CBR variant may be used withone or more additional luciferases, where the luminescence of eachluciferase may be easily resolved using a luminometer withwavelength-discriminating filters enabling the measurement of bothsignals from the same sample. For example, luminescence generated fromthe CBR variant enzyme (between approximately 725-750 nm) and greenCHROMA-LUC™ (approximately 537 nm) can be easily resolved using aluminometer with wavelength-discriminating filters, enabling themeasurement of both signals from the same sample.

In some embodiments, a CBR variant may be used as transcriptionalreporter and paired with either aequorin or a cAMP circularly-permutedfirefly luciferase biosensor, or both simultaneously, to detect multiplepathways in a single sample. In such a system, for example, aequorincould be used for the detection and/or measurement of calcium, thebiosensor for the detection and/or measurement of cAMP, and the CBRvariant for monitoring of downstream gene expression.

The CBR variant may be multiplexed with a luciferase derived from:beetle luciferases, such as firefly (e.g., Photinus pyralis (e.g., Luc2;Promega Corp) or Photuris pennsylvanica luciferases) or a differentclick beetle luciferase (Pyrophorus plagiophthalamus or Pyrearinustermitilluminans), such as green click beetle luciferase (CHROMA-LUC™;Promega Corp.), a bioluminescent decapod, e.g. Oplophorus luciferase,such as Oplophorus gracilirostris luciferases or variants thereof, e.g.,NanoLuc® luciferase (Promega Corporation), as described in U.S. Pat.Nos. 8,557,970 and 8,669,103, and U.S. Patent Publication Nos.2014/0223590 and U.S. Patent Publication No. 2014/0227759, marineorganisms such as cnidarians (e.g., Renilla reniformis luciferase),copepod luciferases, such as Gaussia luciferase, e.g., Gaussia princepsluciferase, Metridia luciferases, e.g., Metridia longa and Metridiapacifica luciferases, Vargula luciferases, e.g., Vargula hilgendorfiiluciferase, Pleuromamma xiphias luciferase, a glowworm luciferase (e.g.Phrixothrix hirtus), and variants, recombinants, and mutants thereof.The CBR variants may be multiplexed with green fluorescent protein(GFP), photoproteins such as Aequorin, obelin, iPhotina, and variants,recombinants, and mutants thereof. For example, if a CBR variant of thepresent invention is used as a functional reporter, then the greenfirefly luciferase or green CHROMA-LUC™ luciferase could be used tocontrol for non-specific effects on genetic regulation or to normalizefor transfection efficiency.

d. Detection of Non-Luminescent Enzymes

The CBR variant and bioluminescence system containing the pro-substratedescribed herein may be used in luminescence-based assays to detect theactivity of a non-luminescent enzyme. The CBR variant and pro-substratemay be contained in an assay reagent for measuring a specific aspect ofcellular physiology, for example ATP to estimate cell viability, orcaspase activity to estimate cellular apoptosis. The CBR variant and thepro-substrate may be added to a sample suspected of containing thenon-luminescent enzyme of interest. The non-luminescent enzyme presentin the sample reacts with the pro-substrate releasing the luciferinsubstrate for the CBR variant to use as a substrate and thusluminescence is generated and measured. In some embodiments, thenon-luminescent enzyme of interest is a reductase, glycosidase,protease, serine proteases, threonine proteases, cysteine proteases,such as caspase-3 and caspase-8, aspartate proteases, glutamic acidproteases, and metalloproteases, peptidase, oxidase, such as a monoamineoxidase enzyme, esterase, cytochrome P450, beta-lactamase, glycosylaseand glutathione transferase protease enzyme, such as a glutathioneS-transferase enzyme. In some embodiments, the pro-substrates, asdescribed herein may be used to detect the non-luminescent enzymes.

e. Bioluminescence Resonance Energy Transfer (BRET)

The CBR variant and bioluminescence system may be used in any method fordetecting ligand-protein and/or protein-protein interactions. In variousembodiments, the CBR variant enzymes may be used to transfer energy toan energy acceptor. One such method is Bioluminescence Resonance EnergyTransfer (BRET). With respect to BRET, energy transfer from abioluminescent donor to a fluorescent acceptor results in a shift in thespectral distribution of the emission of light. This energy transfer mayenable real-time monitoring of protein-protein or ligand-proteininteraction in vitro or in vivo.

In some embodiments, the CBR variant enzymes used in BRET analysis canbe used to determine if two molecules are capable of binding to eachother or co-localize in a cell. For example, a CBR variant enzyme can beused as a bioluminescence donor molecule which is combined with amolecule or protein of interest to create a first fusion protein. Invarious embodiments, the first fusion protein contains a CBR variantenzyme and a protein of interest. In various embodiments, the firstfusion proteins containing the CBR variant enzyme can be used in BRETanalysis to detect protein/protein interaction in systems including butnot limited to cell lysates, intact cells, and living animals. Invarious embodiments, HALOTAG® can be used as a fluorescent acceptormolecule. In some embodiments, HALOTAG® can be fused to a second proteinof interest or to a CBR variant enzyme. For example, a CBR variantenzyme can be fused to HALOTAG®, expressed in cells or animals, andlabeled with a fluorescent HALOTAG® ligand such as HALOTAG® TMR ligand.The fusion can subsequently be excited to fluoresce in the presence of acell-permeant CBR variant enzyme substrate. In some embodiments, BRETmay be performed using CBR variant enzymes in combination withfluorescent proteins, including but not limited to Green FluorescentProtein (GFP), Red Fluorescent Protein (RFP), far red fluorescentprotein, and near infrared fluorescent proteins, such as iRFP andIFP1.4, or fluorescent labels including fluorescein, rhodamine green,Oregon green, Alexa 488, to name a few non-limiting examples. Thefluorescent label may be a fluorescent BRET acceptor that absorbs at theemission of the luciferase (e.g., 650-750 nm), such as IRDye® 800CW,IRDye® 800RS, IRDye® 800 phosphoramidite, IRDye® 750, IRDye® 700DX,IRDye® 700 phosphoramidite, IRDye® 680LT, IRDye® 680RD, IRDy IRDye® e650, 1,1′,3,3,3′,3′-Hexamethylindotricarbocyanine iodide,1,1′-Diethyl-2,2′-dicarbocyanine iodide, 1,1′-Diethyl-4,4′-carbocyanineiodide, 1,4,8,11,15,18,22,25-Octabutoxy-29H,31H-phthalocyanine,2,11,20,29-Tetra-tert-butyl-2,3-naphthalocyanine,2,3,9,10,16,17,23,24-Octakis(octyloxy)-29H,31H-phthalocyanine,2,3-Naphthalocyanine Dye content,2,9,16,23-Tetra-tert-butyl-29H,31H-phthalocyanine,29H,31H-Phthalocyanine β-form, 3,3′-Diethylthiadicarbocyanine iodide,3,3′-Diethylthiatricarbocyanine iodide, 3,3′-Diethylthiatricarbocyanineperchlorate, Aluminum1,8,15,22-tetrakis(phenylthio)-29H,31H-phthalocyanine chloride, Aluminum2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine chloride, Aluminum2,9,16,23-tetraphenoxy-29H,31H-phthalocyanine chloride, Aluminum2,9,16,23-tetraphenoxy-29H,31H-phthalocyanine hydroxide, Aluminumphthalocyanine chloride, Aluminum phthalocyanine hydroxide, Cobalt(II)1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine,Cobalt(II) 2,3-naphthalocyanine, Cobalt(II) phthalocyanine β-form,Copper phthalocyanine-3,4′,4?,4?′-tetrasulfonic acid tetrasodium salt,Copper(II)1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine,Copper(II) 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine,Copper(II)2,3,9,10,16,17,23,24-octakis(octyloxy)-29H,31H-phthalocyanine,Copper(II) 2,3-naphthalocyanine, Copper(II)2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine, Copper(II)phthalocyanine-tetrasulfonic acid tetrasodium salt, Copper(II)phthalocyanine, Copper(II) phthalocyanine β-form, Copper(II)phthalocyanine sublimed grade, Copper(II) phthalocyanine, Dilithiumphthalocyanine, Disodium phthalocyanine, Gallium(III)-phthalocyaninechloride, IR-775 chloride, IR-780 iodide, IR-783, IR-792 perchlorate,IR-797 chloride, Iron(II) phthalocyanine, Iron(III) phthalocyaninechloride, Iron(III) phthalocyanine-4,4′,4″,4′″-tetrasulfonic acid,compound with oxygen monosodium salt hydrate, Lead(II) phthalocyanine,Lead(II) tetrakis(4-cumylphenoxy)phthalocyanine, Magnesiumphthalocyanine, Manganese(II) phthalocyanine, Manganese(III)phthalocyanine chloride, Methylsilicon(IV) phthalocyanine hydroxide,Naphthol Green B, Nickel(II)1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine, Nickel(II)phthalocyanine-tetrasulfonic acid tetrasodium salt, Nickel(II)phthalocyanine, Poly(copper phthalocyanine), Silicon2,3-naphthalocyanine bis(trihexylsilyloxide), Silicon2,3-naphthalocyanine dichloride, Silicon 2,3-naphthalocyaninedihydroxide, Silicon 2,3-naphthalocyanine dioctyloxide, Silicon2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine dihydroxide, Siliconphthalocyanine dichloride, Silicon phthalocyanine dihydroxide, Tin(IV)phthalocyanine oxide, Titanium(IV) phthalocyanine dichloride, Titanylphthalocyanine, Titanyl phthalocyanine, Vanadyl 2,3-naphthalocyanine,Vanadyl3,10,17,24-tetra-tert-butyl-1,8,15,22-tetrakis(dimethylamino)-29H,31H-phthalocyanineDye content 80%, Zinc1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine,Zinc 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine, Zinc2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine, Zinc2,3,9,10,16,17,23,24-octakis(octyloxy)-29H,31H-phthalocyanine, Zinc2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine, Zinc phthalocyanine,or Zinc(II) tetranitrophthalocyanine.

f. Protein Proximity Assays for Live Cells or Lytic Formats

In some embodiments, the CBR variant may be used as a circularlypermuted (CP) or straight split (SS) luminescent enzyme fusion proteinto measure protein proximity. The CBR variant enzyme is permuted orsplit via insertion of a protease substrate amino acid sequence (e.g.,TEV) to generate low bioluminescence. The CBR variant luminescent enzymeis tethered (e.g., via genetic fusion) to a monitor protein. A potentialinteracting protein is tethered (e.g., via genetic fusion) to a protease(e.g., TEV). When the two monitor proteins interact or are in sufficientproximity (e.g., via a constitutive interaction, a drug stimulus or apathway response), the CBR variant enzyme is cleaved to generateincreased bioluminescence activity. The example may be applied tomeasurements of protein proximity in cells or in biochemical assays.

g. Protein Complementation Assays

In some embodiments, the disclosed compounds may be used in othermethods for detecting ligand-protein and protein-protein interactions orproximity, such as the protein complementation assay (PCA) or enzymefragmentation assay. Protein complementation assays (PCA) provide ameans to detect the interaction of two biomolecules, e.g., polypeptides.PCA utilizes two fragments of the same protein, e.g., the CBR variant,that when brought into close proximity with each other can reconstituteinto a functional, active protein. In some embodiments, the PCAutilizing a CBR variant may be used to detect molecular proximity byvirtue of the reconstitution of the CBR variant via the bindinginteraction of enzyme components or subunits. Fragments of the CBRvariant are fused to proteins of interest. If the proteins of interestinteract, the fragments of the CBR variant interact to reconstitute afull-length CBR variant enzyme.

For example, the CBR variant enzyme can be separated into two fragmentsat a site(s) tolerant to separation and each fragment of the separatedCBR variant enzyme can be fused to one of a pair of polypeptides ofinterest believed to interact, e.g., FKBP and FRB. If the twopolypeptides of interest do in fact interact, the CBR variant enzymefragments, for example, then come into close proximity with each otherto reconstitute the functional, active CBR variant enzyme. In someembodiments, the activity of the reconstituted CBR variant enzyme canthen be detected and measured using the disclosed compounds and thecell-permeable substrate. In some embodiments, the split CBR variantenzyme can be used in a more general complementation system similar tolac-Z (Langley et al., PNAS 72:1254-1257 (1975)) or ribonuclease S(Levit and Berger, J. Biol. Chem. 251:1333-1339 (1976)). In someembodiments, a CBR variant enzyme fragment (designated “A”) known tocomplement with another CBR variant enzyme fragment (“B”) can be fusedto a target protein, and the resulting fusion can be monitored vialuminescence in a cell or cell lysate containing fragment B. In someembodiments, the source of fragment B could be the same cell (e.g., ifthe gene for fragment B is integrated into the genome of the cell or iscontained on another plasmid within the cell) or it could be a lysate orpurified protein derived from another cell. In some embodiments, thissame fusion protein (fragment A) could be captured or immobilized usinga fusion between fragment B and a polypeptide such as HALOTAG® capableof attachment to a solid support. In some embodiments, luminescence canbe used to demonstrate successful capture or to quantify the amount ofmaterial captured.

h. Biosensors

The CBR variants may be used as biosensors, which, in the presence ofanother molecule (e.g., one or more molecules of interest), or undercertain conditions, has one or more altered activities. Upon interactingwith a molecule of interest or being subject to certain conditions, thebiosensor undergoes a conformational change or is chemically alteredwhich causes an alteration of the enzyme activity or luminescence, e.g.,specific activity, spectral distribution, or emission kinetics. Forexample, the CBR variant of the present invention, for example acircularly permuted variant, can comprise an interaction domain for amolecule of interest. Alternatively, for example, the CBR variant may becoupled to an energy acceptor, for example a fluorescent protein, andcomprise an interaction domain that alters the efficiency of energytransfer from the enzyme to the energy acceptor. For example, thebiosensor could be generated to detect proteases, kinases, a ligand, abinding protein such as an antibody, cyclic nucleotides such as cAMP orcGMP, or a metal such as calcium, by insertion of a suitable sensorregion into the CBR variant sequence. One or more sensor region can beinserted at the C-terminus, the N-terminus, and/or at one or moresuitable location in the polypeptide sequence, where the sensor regioncomprises one or more amino acids. In the case of a circularly-permutedCBR variant, the sensor region may be inserted between the N- andC-termini of the parent CBR variant. In addition, one or all of theinserted sensor regions may include linker amino acids to couple thesensor to the remainder of the CBR variant polypeptide.

In some embodiments, full-length circularly permuted CBR variant enzymesmay be fused to respective binding partners, e.g., FRB and FKBP, andused in a protein complementation-type assay with the luciferin or novelluciferin derivative. The key difference between the method disclosedherein and traditional protein complementation is that there was nocomplementation, but rather there was dimerization of two full lengthenzymes, e.g., circularly permuted CBR variant enzymes.

Briefly, the circularly permuted reporter proteins similarly configuredfor low activity are fused to both of the fusion protein partners. Forexample, each fusion partner may be linked to identically structured,permuted reporters. Interaction of the fusion partners brought thepermuted reporters into close proximity, thereby allowing reconstitutionof a hybrid reporter having higher activity.

5. SAMPLE

The disclosed CBR variants and/or near-IR bioluminescent systems may beused with samples containing biological components. The sample maycomprise cells and/or tissue. The sample may comprise heterogeneousmixtures of components (including intact cells, cell extracts, celllysates, bacteria, viruses, organelles, and mixtures thereof) or asingle component or homogeneous group of components (e.g., natural orsynthetic amino acid, nucleic acid or carbohydrate polymers, or lipidmembrane complexes). The compounds are generally non-toxic to livingcells and other biological components within the concentrations of use.

The sample may include an animal (e.g., a vertebrate), a plant, afungus, physiological fluid (e.g., blood, plasma, urine, mucoussecretions and the like), a cell, a cell lysate, a cell supernatant, ora purified fraction of a cell (e.g., a subcellular fraction). In certainembodiments, the sample may be a cell. In some embodiments, the samplemay be a live cell. The cell may be a eukaryotic cell, e.g., yeast,avian, plant, insect or mammalian cells, including but not limited tohuman, simian, murine, canine, bovine, equine, feline, ovine, caprine orswine cells, or prokaryotic cells, or cells from two or more differentorganisms, or cell lysates or supernatants thereof. The cells may nothave been genetically modified via recombinant techniques(nonrecombinant cells), or may be recombinant cells which aretransiently transfected with recombinant DNA and/or the genome of whichis stably augmented with a recombinant DNA, or which genome has beenmodified to disrupt a gene, e.g., disrupt a promoter, intron or openreading frame, or replace one DNA fragment with another. The recombinantDNA or replacement DNA fragment may encode a molecule to be detected bythe methods of the invention, a moiety which alters the level oractivity of the molecule to be detected, and/or a gene product unrelatedto the molecule or moiety that alters the level or activity of themolecule. The cells may have been genetically modified via recombinanttechniques.

6. KIT

Kits for using the CBR variant and/or the near-IR bioluminescent systemsare provided herein. Such kits comprise an active CBR variant andluciferin substrate. The kits may further include a buffer andinstructions. The kit components, compositions, and buffers may also bemodified by the addition of suitable components. Suitable kitcomponents, compositions and buffers that may be used in the describedmethods can also be obtained commercially. The different components maycomprise subsets of these parts and may be combined in any way thateither facilitates the application of the invention or prolongs storagelife.

In some embodiments, the kit comprises a separate container comprisinglyophilized luciferase. In some embodiments, the container comprisinglyophilized luciferase further comprises lyophilized luciferin or aderivative thereof that is a luciferase substrate.

One or more reagents may be supplied in a solid form or liquid bufferthat is suitable for inventory storage, and later for addition into thereaction medium when the method of using the reagent is performed.Suitable packaging is provided.

(1) Containers/Vessels

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized luciferase orbuffer that has been packaged under a neutral, non-reacting gas, such asnitrogen. Ampules may consist of any suitable material, such as glass,organic polymers, such as polycarbonate, polystyrene, etc., ceramic,metal or any other material typically employed to hold reagents. Otherexamples of suitable containers include simple bottles that may befabricated from similar substances as ampules, and envelopes, that mayconsist of foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, bottles, syringes, or thelike. Containers may have a sterile access port such as a bottle havinga stopper that can be pierced by a hypodermic injection needle. Othercontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to mix.Removable membranes may be glass, plastic, rubber, etc.

(2) Instructional Materials

The kits may also be supplied with instructional materials. Instructionsmay be printed on paper or other substrate and/or may be supplied as anelectronic-readable medium such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audio tape, etc. Detailed instructions may not bephysically associated with the kit; instead, a user may be directed toan internet web site specified by the manufacturer or distributor of thekit, or supplied as electronic mail.

7. EXAMPLES

The present invention can be utilized as illustrated by the followingnon-limiting examples.

Example 1 Characterization of Near-IR Substrates PBI-4739 and PBI-4813with Ultra-Glo™ Luciferase, QuantiLum® Recombinant Luciferase, andPurified Click Beetle Red Luciferase (CBR)

Materials:

The following were used in the Examples: Ultra-Glo™ Luciferase (PromegaCat. No. E140); QuantiLum® Recombinant Luciferase (Promega Cat. No.E1701); Click Beetle Red Luciferase (CBR; 0.5 mg/mL purified; Promega);Bright-Glo™ assay buffer (Promega Cat. No. E264A); PBI-4739 (Promega—SeeFIG. 1); and PBI-4813 (Promega—See FIG. 1).

Experimental Details.

The substrates, PBI-4813 and PBI-4739, were diluted to 1 mM (finalconcentration) in Bright-Glo™ Assay buffer+1 mM ATP. 50 μL of substratesolution was added to 50 μL of Click Beetle Red, UltraGlo® or QuantiLum®purified enzyme (0.5 mg/mL diluted in DMEM+0.1% Prionex) in triplicate,and then the samples assayed using a Tecan M-1000 plate reader inspectral scan mode.

FIG. 2 provides the spectral data for Click Beetle Red Luciferase withPBI-4813 (A) and PBI-4739 (B), where the spectral maximum was 655 nm forPBI-4813 and 760 nm for PBI-4739. FIG. 3 provides the spectral data forUltraGlo® luciferase with PBI-4813 (A) and PBI-4739 (B), where thespectral maximum was 670 nm for PBI-4813 and 660 nm for PBI-4739. FIG. 4provides the spectral data for QuantiLum® luciferase (firefly) withPBI-4813 (A) and PBI-4739 (B), where the spectral maximum was 680 nm forPBI-4813 and equal to or greater than 700 nm for PBI-4739.

Example 2 Library Screening

A library of Click Beetle Red variants was prepared using the Diversify™PCR Random Mutagenesis Kit (Clontech) according to the manufacturer'sinstructions using pF4Ag-HT7-CBR as a template, i.e., CBR-HALOTAG®fusion protein. The library of variant DNA was cloned into the pF4Ag-HT7(Promega) and transformed into 50 μL KRX competent cells (PromegaCorporation). The cells were grown overnight on LB-ampicillin plates at37° C.

Colonies were picked and grown overnight at 37° C. in 200 μL ofM9-minimal media (1× M9 salts, 0.1 mM CaCl₂, 2 mM MgSO₄, 1 mMThiamine-HCL, 1% gelatin, 0.2% glycerol and 100 μg/mL Ampicillin) inwells of 96-well plates. 10 μL of the overnight culture was diluted into190 μL M9-minimal media and grown overnight at 37° C. 10 μL of thesecond overnight culture was diluted into 190 μL M9-minimal inductionmedia (M9 minimal media+0.05% glucose and 0.02% rhamnose) and grownovernight at 25° C.

The cells were assayed via a robot. Briefly, 25 μL of cell culture wasadded to 25 μL of lysis buffer (50 mM HEPES pH 7.5, 0.3× Passive LysisBuffer (PLB; Promega Corporation), 0.006 U RQ1 DNase1 (PromegaCorporation)) and incubated for 3 min. 50 μL of Bright-Glo™ AssayReagent (Promega Corporation) or PBI-4813 assay reagent (Bright-Glo™Assay buffer+ATP (to 1 mM) and PBI-4813 (to 5 μM)) was added to lysedcells, and luminescence detected on Tecan Genios Pro (Table 1).

TABLE 1 PBI-4813 Bright-Glo ™ Secondary Secondary Sequence # Samplescreen screen Mutation AA 45 91B4 3 1.5 G251S 8 18D5 2.6 1.30 Y252C 3374C2 2.5 3 I389F 34 74F8 2.5 NA I131T, K146E, V285A, N401S 27 46H5 2.41.30 I389F 32 74A8 2.4 2 H218L, D471V 26 44C9 2.2 1.00 F87S, E453K,K484E 1 01 E5 2.1 1.04 L113Q, H218Y 23 29C11 2.1 3.10 Y252C, E501G 919A3 2 1.60 F87S 30 63D9 2 1.10 V431A 16 09G5 1.9 1.80 I131N 29 63D7 1.91.10 T528A 21 15C7 1.8 1.20 C335S 25 40D8 1.8 3.70 N83H 38 75B4 1.73 2.6E253K, T363S 15 16D2 1.7 2.00 E82G, D412G 43 87A4 1.7 2.2 I79V, V394M 4795F4 1.7 1 N211N 4 13F1 1.6 2.60 Y170C, S358P, T363A, K539R 19 17D7 1.61.30 I109V, K484M 46 94F9 1.6 1.5 I409T 20 40B9 1.5 1.80 L228P 36 100D11.5 1.1 V104D, K484R 41 83E 2 1.5 1.2 S444R 42 85 A2 1.5 1.3 F55V, E473A44 90B7 1.5 2.6 V255D 50 100B2 1.5 1.3 M117T, K136N, N156D 7 13G5 1.40.80 E319G 17 12D5 1.4 1.30 F55L,Y496H 39 79G9 1.4 3.2 N130K,V255F 4896H5 1.4 2.5 V455D 3 09G7 1.3 2.70 H34Y, N74S, R280S 5 06G4 1.3 0.60K179S, H218Y, V516A 6 13A3 1.3 0.84 M393K, S444C, K457N

Example 3 Click Beetle Red Mutation Combinations

Some of the mutations identified in the library screen outlined inExample 2 were combined to identify beneficial combinations ofmutations. The mutations were introduced into the Click Beetle Red (CBR)luciferase (SEQ ID NO: 1) using the QuikChange Multi Site-DirectedMutagenesis Kit (Agilent). Variants were cloned, expressed and screenedas described in Example 2. Table 2 and FIG. 5 list the variants andtheir fold change in luminescence over CBR luciferase with the substratePBI-4813 or PBI-4739.

TABLE 2 Sample sequence Sample PBI-4813 PBI-4739 I389F + S444R ATG 10782.48 2.19 I79V + I389F + I309T 11G11 2.47 1.98 G251S ATG 1023 2.28 1.27I370T + I389F + I409T 11A8 2.16 1.81 I79V + I370T + I389F 11C4 1.88 1.55I389F ATG 1032 1.82 1.48 S444R ATG 1020 1.68 1.87 I370T + I389F 1G111.42 1.17 WT WT 1.06 1.05 WT WT 1.05 1.08 WT WT 1.04 1.02 WT WT 1.041.06

Example 4 Variants Tested in Mammalian Cells

Some of the variants identified in Examples 2 and 3 were screened fortheir performance in mammalian cells. DNA was prepared by Plasmid.com.HEK293 and HeLa cells were plated into wells of a 24-well plate at0.05×10⁶ cells/mL and grown overnight at 37° C. with CO₂. 2.2 μg variantDNA was mixed with 80 μL OptiMEM and 6.6 μL FuGENE® HD transfectionreagent (Promega Corporation) and incubated at room temperature for 5min. 25 μL of the DNA mixture was added to each well.

After overnight incubation, the media was removed from the cells and 1mL DPBS (Life Technologies) was added. The cells then underwent afreeze-thaw to generate cell lysates. 50 μL of the lysate was aliquotedinto wells of a 96-deep well plate. 50 μL of assay reagent (20 μM ofPBI-4813 or PBI-4739 in Bright-Glo™ Assay buffer containing 1 mM ATP)was added to the lysates, and luminescence was detected on anImageQuant. Luminescence of the variants was normalized to the averageof two CBR samples (FIG. 6 and FIG. 7).

Example 5 Hydrogen Acceptor Mutagenesis Design

The amino acid differences between CBR and CBG99 contribute to colorchange, such as amino acid changes that have a major contribution,Y224V, H247S, and Q348H, and amino acid changes that have a minorcontribution, I346N and T349S. At position 351, the amino acid isglycine in CBR and CBG99 and arginine in CBG68.

Efficient light emission from some red-shifted luciferin derivatives mayrequire the presence of an amino acid that can serve as a hydrogenacceptor (H-acceptor) at an appropriate position in the active site of aCBR variant. For example, PBI-4739 may require an H-acceptor in thevicinity of its hydroxyl group. The computational experiments describedbelow were performed using Discovery Studio software (Accelrys). Ahomology model of CBR was generated based on an X-ray structure templateof the related Luciola cruciata luciferase complexed with DLSA (ProteinData Bank accession 2D1S). The PBI-4739 ligand was manually docked intothis model to match the position of the DLSA ligand in 2D1S, and aminoacid side chains within 5 Å of PBI-4739 were energy minimized. Moleculardynamics simulation was performed on CBR amino acid side chains within 5Å of PBI-4739. Representative output conformations were used to examinein silico which CBR mutations (amino acid replacements) might providethe desired H-acceptor.

A first group of mutations was identified based on restructuring anetwork of interacting side chains. The targeted positions were in thevicinity of the PBI-4739 hydroxyl and at least partially exposed tosolvent. Based on an alignment of representative beetle luciferases(firefly, click beetle, glowworm), it was determined that the sidechains at these positions are conserved (E308), mostly conserved (R334and T226) or variable (G351). Various combinations of side chains atthese positions allowed formation of a stabilizing H-bond network thatshields the ligand from solvent and bridges multiple secondary structureelements. The mutation combinations below were intended to providedifferent H-bond networks while at the same time recruited one of theside chains as an H-acceptor for interaction with hydroxyl of PBI-4739:

G351R+R334 (E, Q, D, N, H, S, T, C, Y)

G351K+R334 (E, Q, D, N, H, S, T, C, Y)

G351K+T226N+R334 (E, Q, D, N, H, S, T, C, Y)

G351E+E308R+R334 (E, Q, D, N, H, S, T, C, Y)

E308R+R334 (E, Q, D, N, H, S, T, C, Y)

T226N+R334 (E, Q, D, N, H, S, T, C, Y)

A second group of mutations was suggested based on targeting individualpositions that can accommodate a variety of H-acceptors. Preference wasgiven to substitutions that would not be accessible by a randommutagenesis approach, i.e. not accessible by single base substitutions:

T226 (N, Q, E, D, H, C, Y)

C310 (Q, E, N, D, H)

Q348 (H, E)

S281 (N, Q, H, Y)

Example 6 Hydrogen Acceptor Mutagenesis Variant Screen

A. Mutagenesis.

Hydrogen acceptor mutagenesis was performed using the QuikChange MultiSite-Directed Mutagenesis Kit (Agilent) as previously described usingdegenerate oligos specific to the amino acids listed below. Thefollowing variants were cloned and expressed as described in Example 2:

T226 (Q,N,E,D,H,C,Y)

C310 (Q,E,N,D,H)

S281 (Q,N,H,Y)

Q348 ((H,E)

G351R+R334 (E,Q,D,N,H,S,T,C,Y)

G351K+R334 (E,Q,D,N,H,S,T,C,Y)

G351+226N+R334 (E,Q,D,N,H,S,T,C,Y)

G351E+E308R+R334 (E,Q,D,N,H,S,T,C,Y)

E308R+R334 (C,Y)

T226N+R334(E,D,S,T,C,Y)

B. Primary Screen.

1. One plate of clones was picked for each of the 10 sets of amino acidcombinations described. These plates were then sequenced and assayed aspreviously described.

2. Cell lysates were prepared from cells containing the variants listedin Table 3 by diluting 100 μL of induced culture with 100 μL of lysisbuffer (0.3×PLB, 0.006 U RQ DNAse1) using a Tecan liquid handling robot.50 μL of each cell lysate was assayed with either PBI-4813 or PBI-4739(20 μM in Bright-Glo™ assay buffer containing 1 mM ATP), andluminescence detected on an ImageQuant CCD imager. Luminescence of thevariants was normalized to CBR luciferase (FIG. 8).

C. Secondary Screen.

1. Hits from the primary screen were selected and then processed in asecondary screen. Each sample was assayed in quadruplicate using thesame assay method described above for the primary screen.

2. The variants were assayed as described above in for the primaryscreen. Luminescence of the variants was normalized to CBR luciferase(Table 3).

TABLE 3 Sample sequence Sample PBI-4813 PBI-4739 351R + 334S 5D5 0.7 3.3226N + 351K + 334S 7D1 0.5 3.1 226N + 334N 10B2 0.5 2.9 R334S 5B9 0.62.9 351K + 334S 6F9 0.7 2.9 226N + 334S 10F2 0.4 2.8 351K + 334S 6A1 0.62.7 351K + 334Q 6G7 0.9 2.4 351R 5E 2 0.7 2.2 226H 1F9 1.6 1.9 351R +334H 5C4 1.6 1.8 226G 1B3 1.3 1.7 351K + 334E 6G3 0.8 1.7 348E 4D1 1.11.7 226C 1H3 1.2 1.6 226Y 1A12 1.5 1.6 S281N S281N 0.9 1.5 S281Q S281Q0.9 1.5 226N 1G2 1.3 1.5 226N 7G4 1.2 1.5 351K + 334K 6C1 1.3 1.4 226Q1D5 1.1 1.4 226N 1F8 1.1 1.4 348H 4H1 0.6 1.3 WT WT 1.0 1.1 WT WT 1.01.1 WT WT 1.1 1.1 WT WT 1.0 1.0

Example 7 Combinations of H-Acceptor Mutagenesis and Library ScreenMutants

Combinations of the Hydrogen acceptor mutants identified in Example 6were designed and prepared using the QuikChange Multi Site-DirectedMutagenesis Kit (Agilent). Variants were cloned and expressed asdescribed in Example 2 except the template was pF4Ag-CBR. Variants werescreened as described in Example 6. Luminescence of the variants usingluciferin (“D-LH2”), PBI-4813, and PBI-4739 was normalized to CBRluciferase (Table 4 and FIG. 9).

TABLE 4 Sequence D-LH2 (luciferin) PBI-4813 PBI-4739 226N + 351K + 334S1.2 0.5 2.4 (685 template) ATG 685 + 334S 1.0 0.4 2.6 389F + 444 (685template) 0.8 2.0 1.5 334S + 334K (685 template) 1.2 0.6 2.9 ATG 685 1.01.0 1.0 389F (343 template) 1.0 1.5 1.1 389G (343 template) 0.6 1.7 1.9ATG 685 + 334S + 351R 1.2 0.6 3.2 389S (343 template) 0.9 1.5 1.5 389T(343 template) 1.1 1.3 1.3 ATG 343 1.0 1.0 1.0 334S + 351R + 389F 0.70.5 2.4 (343 template) 334S + 351R + 389G 0.4 0.7 2.4 (343 template)334S + 351R + 389F + 0.5 0.7 2.1 409T (343 template) 334S + 351R +389F + 0.5 0.7 1.9 409T (343 template) 334S + 389F + 444R 0.5 0.5 2.3(343 template) 334S + 389F (343 template) 0.7 0.5 2.4 334S + 389F (343template) 0.7 0.4 2.4 I389G (343 template) 0.5 1.7 2.0 389F + 409T (343template) 0.7 1.7 1.4 389F + 444T (343 template) 0.7 1.7 1.4 251S +389F + 444R 0.6 3.4 1.6 (343 template) 251S + 389F + 444R 0.5 3.2 1.4(343 template) 251S + 389G + 444R 0.1 2.2 1.2 (343 template)

Example 8 Evaluation of Clone 1230 and 1240

Variant clones identified as ATG 1230 (CBR+389F+444R+251S) and ATG 1240(CBR+334S+351R) were further evaluated for their luminescence, spectralproperty, Km and live cell kinetics with either PBI-4813 or PBI-4739.These variants were cloned and expressed as described in Example 2except the template was pF4Ag-CBR, i.e., no HALOTAG®.

A. Luminescence with PBI-4813 and PBI-4739.

Cell lysates of these variant clones were prepared and assayed forluminescence as described in Example 6. Luminescence was detected on aGloMax® Discover equipped with a red-sensitive photomultiplier tube(PMT) and compared to CBR luciferase (Table 5 and FIG. 10).

TABLE 5 PBI- 4813/ PBI PBI- PBI- Clone 4813 stdev cv 4739 stdev cv 4739CBR 98,556 605.016 0.6% 17,431 605.016 3% 5.7 ATG 310,342 1133.41 0.4%26,595 1133.41 4% 11.7 1230 ATG 71,299 1614.31 2.3% 37,169 1614.31 4%1.9 1240

B. Spectral Measurements.

Purified ATG 1230 and ATG 1240 enzyme were prepared. CBR was diluted1:10 in 0.3×PLB+0.1% Prionex. The enzymes were assayed in triplicate byadding 50 μL of enzyme to 50 μL of assay reagent (20 μM PBI-4813 orPBI-4739 in Bright-Glo™ Assay buffer containing 1 mM ATP). Luminescencewas detected on a Tecan-M1000 using spectral scan mode (FIG. 11). UsingPBI-4813 as substrate, ATG-1240 had a spectral max at about 725 nm whileCBR and ATG-1230 had a spectral max of about 650 nm. Using PBI-4739 assubstrate, ATG-1240 had a spectral max at about 750 nm while CBR andATG-1230 had a spectral max of about 760 nm.

C. Km Titration.

Cell lysates of ATG 1230 and ATG 1240 expressed in HEK293 cells wereprepared as described in Example 4. The cell lysates were then diluted1:10 in 0.3×PLB+0.1% Prionex. Dilutions (2×) of each substrate (PBI-4813or PBI-4739) were prepared in Bright-Glo™ Assay buffer containing 1 mMATP. 50 μL of diluted cell lysate was assayed with 50 μL of eachsubstrate assay solution. Luminescence was detected on the GloMax®Discover (FIG. 12). FIG. 12A and FIG. 12B show the relative Vmax (RLU)and Km (μM) values for ATG 1230 and ATG 1240 lysates using PBI-4813 andPBI-4739 as substrates. Because the enzyme concentration is not knownfor the lysates of for live cell titrations, Vmax as expressed as avelocity is not possible. Instead, the relative Vmax calculates themaximum brightness for each lysate with different substrates. The Km forPBI-4813 with CBR in the lysates was about 30× lower than CBR withD-luciferin. The Km for PBI-4813 with ATG1240 was about 5× lower thanCBR with D-luciferin.

D. Live Cell Kinetics.

HEK293 cells were plated into wells of a 96-well plate at 10,000cells/well for the kinetic read and 5,000 cells/well for luminescencedetection. Cells were transfected (n=24 for each sample) using 17 μg ofeach DNA (ATG 1230 or ATG 1240) in a total of 776 μL of OptiMEM. 50 μLof FuGENE® HD (Promega Corporation) was then added, and the samples wereincubated for 10 min at room temperature. 5 μL of DNA complex was addedto 12 wells/clone to each plate. Plates were then incubated overnight at37° C. with CO₂. Cells were assayed by first removing the growth mediaand replacing it with CO₂ independent media+0.5% FBS containing dilutedsubstrate (100 μM of PBI-4813, 1 mM of PBI-4739, or 3 mM ofD-luciferin). For the kinetic reading, the plate was incubated at roomtemperature for 5 min, and the kinetics were measured for 150 min at 37°C. on a GloMax® Discover (FIG. 13). For luminescence detection, theplate was incubated in a CO₂ incubator for 90 min, and luminescence wasdetected on a GloMax® Discover (FIG. 14).

HeLa cells were plated at 20,000 cells/well into wells of 96-well whiteassay plates and grown overnight. A transfection mixture was madecontaining 13 μg of CBR DNA and 40 μL FuGENE® HD in a total volume of620 μL OptiMEM. The mixture was incubated at room temperature for 10min. The cells were then transfected, in triplicate, with 5 μL oftransfection complex and grown overnight.

The transfected cells were then assayed. 3× serial dilutions ofD-luciferin starting at 3 mM in CO₂ independent media+0.5% FBS, and 3×serial dilutions of PBI-4813 starting at 0.3 mM were prepared. Thegrowth media from the transfected cells was removed and replaced withone of the 3× serial diluted substrate containing media. The cells wereincubated for 60 min at 37° C. Luminescence was detected on aGloMax®-Multi+ luminometer (FIG. 21A and FIG. 21B). For CBR in livecells, the Km for PBI-4813 was about 20 times lower than the Km forD-luciferin.

HEK293T cells were plated at 15,000 cells/well into wells of 96-wellwhite assay plates and grown overnight. A transfection mixture was madecontaining 13 μg of CBR DNA and 40 μL FuGENE® HD in a total volume of620 μL OptiMEM. The mixture was incubated at room temperature for 10min. The cells were then transfected, in triplicate, with 5 μL oftransfection complex and grown overnight.

The transfected cells were then assayed. 2× serial dilutions ofD-luciferin starting at 2 mM in CO₂ independent media+0.5% FBS, and 2×serial dilutions of PBI-4813 and PBI-4739 starting at 1 mM wereprepared. The growth media from the transfected cells was removed andreplaced with one of the 3× serial diluted substrate containing media.The cells were incubated 60 min at 37° C., and the cells were imaged ona ImageQuant CCD imager (FIG. 22). For CBR, the Km of PBI-4739 was about5 times lower relative to D-luciferin while the Km of PBI-4813 was about23 times lower relative to D-luciferin.

HEK293T cells were plated at 15,000 cells/well into wells of 96-wellwhite assay plates and grown overnight. A transfection mixture was madecontaining 6.6 μg of CBR or ATG-1240 DNA and 20 μL FuGENE® HD in a totalvolume of 310 μL OptiMEM. The mixture was incubated at room temperaturefor 10 min. The cells were then transfected, in triplicate, with 5 μL oftransfection complex and grown overnight.

The transfected cells were then assayed. 2× serial dilutions of PBI-4813and PBI-4739 starting at 2 mM in CO₂ independent media+0.5% FBS wereprepared. The growth media from the transfected cells was removed andreplaced with one of the serial diluted substrate containing media. Thecells were incubated 60 min at 37° C., and luminescence detected on amodified GloMax® Discover luminometer containing a red shifted PMT (seeFIG. 23A and FIG. 23B). The Km of ATG 1240 was about 5 times higher thanthe Km of CBR with PBI-4813. The Km of ATG 1240 was about 3 times higherthan the Km of CBR with PBI-4739.

Example 9 Secondary Library Screen Using ATG 1240 as a Template

A random clone library was prepared as described in Example 2 using ATG1240 as a template. The variants were then cloned into pF4Ag (no HT7).The library was screened as follows (50 plates total):

i. Primary Screen.

Cells were grown as described in Example 2. Cell lysates were preparedby diluting 100 μL of induced culture with 100 μL of lysis buffer(0.3×PLB+0.006 U RQDNAse1) using a Tecan liquid handling robot. 50 μL ofeach cell lysate was then assayed with either PBI-4813 or PBI-4739. (20μM substrate in Bright-Glo™ assay buffer containing 1 mM ATP).Luminescence was detected on a GloMax® Discover.

ii. Secondary Screen.

Cells from each hit in the primary screen were streaked onto new platesand grown overnight. Colonies were picked in quadruplicate and cultureswere grown as described in Example 2. 50 μL of each cell lysate wasassayed with 50 μL of BrightGlo™ assay buffer containing 1 mM ATP and 30μM PBI-4813 or 50 μM PBI-4739. Luminescence was detected on a GloMax®Discover Multimode Detection System (Table 6). The variants in thislibrary were identified as having on average between 2-3 mutations pergene.

TABLE 6 Sequence Sample PBI-4813 PBI-4739 S51N + I119F 23E 4 2.5 5.0I119T + N329D + N400D 5C4 1.2 1.5 M393L 14B12 1.1 1.5 R4H + I229V 8D61.6 1.4 D352N 2C11 1.4 1.3 H16Q + M73T + I89V + 8A8 1.4 1.3 I109N +E489V D47E, I109N, G200G 48A2 1.22 1.26 L537W 16E 8 1.7 1.2 V186A +I439V 20D7 1.9 1.2 L350P, Q535H 38F8 1.34 1.17 I439V 3C2 1.7 1.2 F420F,V503M 32C8 1.29 1.16 K337E, V431A 48A4 1.56 1.15 I119F, G251I, I346N30A12 0.69 1.12 WT ATG-1240 1.2 1.1 L537W 43 E8 1.12 1.09 M73K, Y508C49F9 1.19 1.08 L537W 29H1 1.04 1.07 N156K + Q445H 4D11 1.4 1.1 WTATG-1240 1.01 1.05 Y422C + E531G 2D4 1.4 1.0 WT ATG-1240 1.0 1.0 WTATG-1240 0.9 1.0 WT ATG-1240 1.01 0.99 WT ATG-1240 1.02 0.99 I124V,G225S 40G2 0.97 0.97 WT ATG-1240 0.97 0.97 WT ATG-1240 0.9 0.9 K72E,N133D, F144L, I409T 49E 3 1.29 0.86 Y52C 35H1 1.39 0.82 E437G, I390I43C10 0.99 0.80 E437G, I390I 45H2 0.95 0.79 V234A, V467A 46H12 0.99 0.67

Example 10 Insertion Mutagenesis: Engineering the Click BeetleRed-Luciferin Binding Pocket

The goal was to significantly increase the light emission from ClickBeetle Red (CBR) mutant(s) with the red-shifted substrates, e.g.,PBI-4739 (more red and less soluble), PBI-4813 (brighter, possiblesubstrate inhibition). The previously described random mutagenesis andsite-directed mutagenesis yielded about 3-fold improvement in lightemission.

Approach 1 was to perform cassette mutagenesis of contiguous residuestretches in the vicinity of luciferin ligand binding. Approach 2 was toattempt to expand the luciferin ligand binding pocket by insertingsingle Ala residues at specific sites.

Suitable insertion sites were identified by visual inspection of thepreviously generated 3D structure model of CBR with PBI-4739, asdescribed in Example 5. Additional information on sites that tolerateinsertions/deletions was obtained from an alignment of diverse beetleluciferase protein sequences (e.g., firefly, click beetle, and glowworm)retrieved from the public GenBank and UniProt databases. Furtherinformation was obtained from superimposing 3D structures of luciferases(e.g. firefly luciferase with ligand, PDB accession 2D1S) and relatednon-luciferases (e.g. CoA-ligase with ligand, PDB accession 3N12) andexamining how sequence changes such as insertions/deletions allowsecondary structure elements move to accommodate different ligands inthe binding pocket.

Based on combined analyses of the 3D structures and protein sequencealignment, several sites in CBR were identified as targets for insertionmutagenesis. Single Ala residues were inserted after these positions(bolded and underlined in FIG. 18) in CBR.

Variant constructs generated by the insertion mutagenesis were preparedby Gene Dynamics. Cells from each variant clone were grown overnightonto LB-ampicillin plates, and colonies picked in quadruplicate andgrown in cultures as described in Example 6. Cell lysates were preparedas previously described, and 50 μL of cell lysate was assayed with 50 μLof Bright-Glo™ assay buffer containing 1 mM ATP and 30 μM PBI-4813 orPBI-4739. Luminescence was detected on a GloMax® Discover and normalizedto CBR (Table 7).

TABLE 7 Neutral insertion mutations - Note: (“A” inserted at designatedposition) Sample PBI-4813 PBI-4739 38 0.9 0.8 39 0.9 1.0 40 0.9 1.0 1440.9 0.8 145 0.9 0.9 146 1.0 0.9 147 1.0 1.1 171 0.9 0.9 173 1.0 0.9 2271.0 0.9 264 0.6 1.1 265 0.8 1.1 350 1.1 0.8 351 1.2 1.0

Example 11 Recombination of ATG 1240 Library Mutants

Combinations of the ATG 1240 library mutants identified in Example 9were designed and prepared using DNA shuffling (Stemmer (1994) PNAS USA91:10747-10751). Briefly, a library of mutant genes was created via DNAshuffling. The mutant genes were then cloned into the pF4Ag vector andscreened as described in Example 6. Luminescence was detected on aGloMax® Discover Multimode Detection System and normalized to ATG-1240luciferase (Table 8 and FIG. 15).

TABLE 8 Sample Sequence PBI-4813 PBI-4739 D-LH2 5G5 S51N + S444R 4.0 3.82.8 8D10 S51N, L113Q, S334R, 6.2 1.8 4.7 R351G 18C9 G251S + S334R + 3.20.8 0.8 I389F + insert A 351 3B9 R4H + K377E + 1.9 1.6 1.5 N400D + I439V8C10 I119T 4.5 1.6 3.7 9F10 D352N + S479T 1.5 1.5 1.3 11D10 R4H + insertA 354 1.8 1.2 1.5 17A4 S334R + R351G + 1.7 1.2 0.7 I389F 10A7 I119F +G251S + 2.7 1.0 0.4 S334R + M393L 18C9 S334R + R351G + 3.2 0.8 0.5 V431A4H12 I229V + S334R + 2.5 0.8 0.8 R351G + K377E + V431A 7G5 G251S +S334R + 3.7 0.7 0.9 I389F 10 E5 V186A + G251S + 3.1 0.6 0.5 S334R +R351G + I439V 6D7 R4H, S334R, R351G, 2.6 0.6 1.3 I389F, S444R 8A1V186A + S334R + 2.2 0.5 0.7 R351G + I389V + V431A 4C8 I119T + G159D +3.2 0.5 0.3 S334R + G251S + R355G + I389F 1E 2 I119T + S334R + 2.2 0.50.7 R351G + I389F 10C11 S51N + I119T 1.2 1.3 0.5

Example 12 Live Cell Substrate Titration

HEK293 cells were plated into wells of a 96-well plate at 5,000cells/well and grown overnight. Cells were then transfected (n=24 foreach sample) using 17 μg of ATG 1240 DNA in a total of 776 μL ofOptiMEM. 50 μL of FuGENE® HD (Promega Corporation) was then added, andthe samples incubated for 10 min at room temperature. 5 μL of DNAcomplex was added to18 wells/sample to each plate. Plates were thenincubated overnight at 37° C. with CO₂.

Cells were assayed by first removing the growth media and replacing itwith CO₂ independent media+0.5% FBS containing 2× diluted substrate(PBI-4813 or PBI-4739). 100 μL of each diluted substrate was added intriplicate to the transfected cells and incubated for 60 min.Luminescence was detected on a GloMax® Discover (FIG. 16).

Example 13 Mimicry of Tissue Attenuation Using Long Pass Filters

This example demonstrates how much light is produced in cellstransfected with Luc2 (Photinus pyralis luciferase) and assayed withD-luciferin compared to ATG 1240 assayed with D-luciferin, PBI-4813 orPBI-4739 using various long pass cut-off filters.

A transfection complex was prepared by diluting ATG 1240 DNA or Luc2 DNA(pF4Ag-Luc2) to 100 ng/μL, and then 2× serial dilutions prepared in 50ng/μL pGEM carrier DNA. 40 μL of the diluted DNA was the mixed with 160μL phenol red free OptiMEM and 16 μL of FuGENE® HD and incubated for 20min. 5 μL of each transfection complex was added to wells of a 96-wellblack assay plates and then 100 μL of diluted HEK293T cells (200,000/mLin DMEM+10% FBS) was added. The cells were grown overnight at 37° C.with CO₂.

To assay the cells, 4 mM D-luciferin, 2 mM PBI-4813 and 2 mM PBI-4739substrate solutions were prepared in DMEM+10% FBS. 100 μL of theD-luciferin solution was added to cells expressing Luc2 and ATG 1240,and 100 μL of PBI-4813 or PBI-4739 solution was added to ATG 1240expressing cells. Samples were incubated for 10 min at 37° C., andluminescence detected on GloMax® Discover (heated to 37° C.) with nofilter, 610 nm Long Pass (LP) filter, 665 nm LP filter, 695 nm LPfilter, 720 nm LP filter or 760 nm LP filter.

These results in FIG. 17 suggest that ATG 1240 assayed with D-luciferinmay be brighter than with PBI-4739 even when light is attenuated withlong pass filters. PBI-4813 and D-luciferin are comparable at thegreatest light attenuation. Note that the Km for PBI-4813 was more than10 times less than for D-luciferin in live cell. This could be anadvantage for PBI-4813 in live animals.

Example 14 Codon Optimization

600,000 cells (HEK293, 3T3, or CHO cells) in 3 mL were plated in 6 wellplates. Briefly, for each cell type grown in t-75 flasks, media wasremoved from the t-75 flask and the cells were washed with DPBS. 3 mL ofTrypsin was added to the cells and the cells were incubated for 3 min.10 mL of growth media (DMEM+10% FBS for HEK293T and 3T3, Ham F12+10% FBSfor CHO) were added. Cells were centrifuged at 500 rpm for 5 min. Thecells were counted and diluted to 200,000 per mL. 3 mL of cells wereadded to each well of a six well plate (i.e., 600,000 cells).

Protocol for transfection of HEK293 cells grown in 3,000 μL of medium in6-well plates using a FuGENE® HD:DNA ratio of 3.0:1. This protocol willprepare sufficient DNA/FuGENE® HD reagent to transfect 3 wells at 3000μL/well.

Cell plating. HEK293 cells were plated the day before transfection at adensity of 5×10⁵ cells per well of a 6-well plate in 3 ml of completegrowth medium (DMEM+10% Fetal Bovine Serum).

Complex preparation. 465 μL of 0.020 μg/μL plasmid solution wereprepared in OptiMEM, OptiPro, or sterile deionized water. Briefly, 9.9μg of test DNA was added in 465 μl total volume. 10 ng of Nanoluc DNAwas added to each reaction (“ATG42”) for normalization. 30 μl of FuGENE®HD reagent was added and mixed carefully by pipetting (15 times) or byvortexing briefly. The mixture incubated for 5 to 10 min at roomtemperature. 150 μl of complex was added per well to the cells, andmixed thoroughly. The Reagent:DNA ratio may range from 2.5:1.0 to3.5:1.0. The recommended Reagent:DNA ratio for HEK293 cells was 3.0:1 at3.0 μg DNA per well.

Re-plate cells: Growth media was removed and the cells were washed with1 ml of DPBS. 3 ml of growth media was added and the cells werecentrifuged at 500 rpm. For each sample, the pellets were re-suspendedin 1 ml of media and the cells were counted. The HEK cells had verylittle pellet and the cell counts were below 100,000/ml. See Table 9 fordilution of 3T3 and CHO cells. 100 μL of each sample (n=16) were platedin a 96 well plate. One plate was used for each cell type.

TABLE 9 Construct Cells/mL*10E6 Volume cells (μL) Volume media (mL) 3T3ATG 343 1.4 286 1.7 ATG 1240 1.2 333 1.7 ATG 1929 1.5 267 1.7 ATG 19441.1 364 1.6 ATG 1945 1.7 235 1.8 CHO ATG 343 1.3 308 1.7 ATG 1240 0.8500 1.5 ATG 1929 0.6 667 1.3 ATG 1944 0.72 556 1.4 ATG 1945 0.75 533 1.5

Assay: Bright-Glo assay buffer was reconstituted with Bright-Glo assaysubstrate. 500 μL of furimazine was added to 25 ml of NanoGlo Assaybuffer. 100 μL of reconstituted Bright-Glo was added to each sample(n=8) and 100 μL of NanoGlo was added to the other samples (n=8) andread on GloMax®-Multi+.

Legend/background information for codon optimization experiments:

Variant 343=CBR sequence (“ATG 343”)

Variant 1240=CBR sequence+R334S/G351R (“ATG 1240”)

Variant 1929=CBR sequence+R334S/G351R (codon optimized for RNA structureand codon usage in rodents) (“ATG 1929”)

Variant 1944=CBR sequence+R334S/G351R (codon optimized for RNA structureand codon usage in CHO cells) (“ATG 1944”)

Variant 1945=CBR sequence+R334S/G351R (codon optimized for RNA structureand codon usage in mouse lung and liver cells) (“ATG 1945”)

While the present invention is described in connection with what ispresently considered to be the most practical and preferred embodiments,it should be appreciated that the invention is not limited to thedisclosed embodiments, and is intended to cover various modificationsand equivalent arrangements included within the spirit and scope of theclaims. Modifications and variations in the present invention may bemade without departing from the novel aspects of the invention asdefined in the claims. The appended claims should be construed broadlyand in a manner consistent with the spirit and the scope of theinvention herein.

For reasons of completeness, various aspects of the invention are setout in the following numbered clauses:

Clause 1. An isolated polynucleotide encoding a click beetle redluciferase (CBR) variant polypeptide having at least 80% amino acidsequence identity to SEQ ID NO: 1 and comprising at least one amino acidsubstitution at a position corresponding to position 4, 16, 34, 47, 51,52, 55, 72, 73, 74, 79, 82, 83, 87, 89, 104, 109, 113, 117, 119, 124,130, 131, 133, 136, 144, 146, 156, 159, 170, 179, 186, 200, 211, 218,224, 225, 226, 228, 229, 234, 247, 251, 252, 253, 255, 280, 281, 285,308, 309, 310, 319, 329, 334, 335, 337, 346, 348, 349, 350, 352, 354,355, 358, 363, 370, 377, 390, 393, 394, 400, 401, 409, 412, 420, 422,431, 437, 439, 444, 445, 453, 455, 467, 471, 473, 479, 484, 489, 496,501, 503, 508, 516, 528, 531, 535, 537, 539, or combination thereof, ofSEQ ID NO: 1, wherein the variant CBR polypeptide has at least one ofenhanced luminescence, altered light emission wavelength, alteredsubstrate specificity, or a combination thereof, as compared to a CBRpolypeptide of SEQ ID NO: 1.

Clause 2. The isolated polynucleotide of clause 1, wherein the CBRvariant polypeptide further comprises at least one amino acidsubstitution at a position corresponding to position 351, 389, 457, orcombination thereof, of SEQ ID NO: 1.

Clause 3. The isolated polynucleotide of clause 1 or 2, wherein the CBRvariant polypeptide comprises a substitution corresponding to at leastone of R4H, H16Q, H34Y, D47E, S51N, Y52C, F55L/V, K72E, I79V, M73K/T,N74S, E82G, N83H, F87S, I89V, V104D, I109N/V, L113Q, M117T, I119F/T,I124V, N130K, I131N/T, N133D, K136N, F144L, K146E, N156D, N156K, G159D,Y170C, K179S, V186A, G200G, N211N, H218L/Y, G225S, T226C/G/H/N/Q/Y,L228P, I229V, V234A, G251S, G251I, Y252C, V255D/F, E253K, R280S,S281N/Q, V285A, I309T, E319G, N329D, R334E/Q/H/S/N/K, C335S, K337E,I346N, Q348H/E, L350P, G351K/R, D352N, R355G, S358P, T363A/S, I370T,I389F/G/S/V, I390I, M393K/L, V394M, N400D, N401S, I409T, D412G, F420F,Y422C, V431A, E437G, I439V, S444C/R/T, Q445H, E453K, V455D, K457N,D471V, E473A, S479T, K484E/M/R, E489V, Y496H, E501G, V503M, Y508C,V516A, T528A, E531G, Q535H, L537W, K539R, or combinations thereof, orSEQ ID NO: 1.

Clause 4. The isolated polynucleotide of any one of clauses 1-3, whereinthe CBR variant polypeptide comprises an amino acid substitution at aposition corresponding to position 389, 444, and 251 of SEQ ID NO: 1.

Clause 5. The isolated polynucleotide of any one of clauses 1-4, whereinthe amino acid substitutions comprise I389F, S444R, and G251S.

Clause 6. The isolated polynucleotide of any one of clauses 1-5, whereinthe CBR variant polypeptide comprises an amino acid substitution atpositions corresponding to positions 334 and 351 of SEQ ID NO: 1.

Clause 7. The isolated polynucleotide of any one of clauses 1-6, whereinthe amino acid substitutions comprise R334S and G351R.

Clause 8. The isolated polynucleotide of any one of clauses 1-7, whereinthe CBR variant polypeptide further comprises an amino acid substitutionat a position corresponding to positions 51 and 444 of SEQ ID NO:1.

Clause 9. The isolated polynucleotide of any one of clauses 1-8, whereinthe amino acid substitutions comprise S51N and S444R.

Clause 10. The isolated polynucleotide of clause 1, wherein the CBRvariant polypeptide comprises an amino acid polypeptide of SEQ ID NO: 2,SEQ ID NO: 3, or SEQ ID NO: 4.

Clause 11. The isolated polynucleotide of any one of the precedingclauses, wherein the CBR variant polypeptide has enhanced luminescencecompared to a CBR polypeptide of SEQ ID NO: 1.

Clause 12. The isolated polynucleotide of clause 11, wherein the CBRvariant polypeptide has enhanced luminescence when a luciferin isutilized by the CBR variant polypeptide to generate luminescence.

Clause 13. The isolated polynucleotide of clause 11, wherein the CBRvariant polypeptide has enhanced luminescence when a luciferinderivative is utilized by the CBR variant polypeptide to generateluminescence.

Clause 14. The isolated polynucleotide of clause 13, wherein theluciferin derivative comprises:

Clause 15. The isolated polynucleotide of clause 14, wherein the CBRvariant polypeptide has at least 2 fold increase in luminescencecompared to a CBR polypeptide of SEQ ID NO: 1.

Clause 16. The isolated polynucleotide of clause 14, wherein the CBRvariant polypeptide has at least 4 fold increase in luminescencecompared to a CBR polypeptide of SEQ ID NO: 1.

Clause 17. The isolated polynucleotide of any one of the precedingclauses, wherein the CBR variant polypeptide has altered light emissionspectra compared to a CBR polypeptide of SEQ ID NO: 1.

Clause 18. The isolated polynucleotide of clause 17, wherein the CBRvariant polypeptide is able to emit light at a longer wavelength when aluciferin is utilized by the CBR variant polypeptide to generateluminescence.

Clause 19. The isolated polynucleotide of clause 17, wherein the CBRvariant polypeptide is able to emit light at a longer wavelength when aluciferin derivative is utilized by the CBR variant polypeptide togenerate luminescence.

Clause 20. The isolated polynucleotide of clause 19, wherein theluciferin derivative comprises:

Clause 21. The isolated polynucleotide of clause 20, wherein if theluciferin derivative comprises

the CBR variant polypeptide emits light having a shift in spectralmaximum of at least about 1 nm to at least about 100 nm relative to thelight produced by the CBR polypeptide of SEQ ID NO: 1.

Clause 22. The isolated polynucleotide of clause 21, wherein the CBRvariant polypeptide emits light having a spectral maximum between about650 nm to about 800 nm.

Clause 23. The isolated polynucleotide of clause 21, wherein the CBRvariant polypeptide emits light having a spectral maximum between about725 nm to about 775 nm.

Clause 24. The isolated polynucleotide of clause 21, wherein the CBRvariant polypeptide emits light having a spectral maximum of about 750nm.

Clause 25. The isolated polynucleotide of clause 20, wherein if theluciferin derivative comprises

the CBR variant polypeptide emits light having a shift in spectralmaximum of at least about 1 nm to at least about 100 nm relative to thelight produced by the CBR polypeptide of SEQ ID NO: 1.

Clause 26. The isolated polynucleotide of clause 25, wherein the CBRvariant polypeptide emits light having a shift in spectral maximum of atleast about 75 nm relative to the light produced by the CBR polypeptideof SEQ ID NO: 1.

Clause 27. The isolated polynucleotide of clause 25, wherein the CBRvariant polypeptide emits light having a spectral maximum between about650 nm to about 800 nm.

Clause 28. The isolated polynucleotide of clause 25, wherein the CBRvariant polypeptide emits light having a spectral maximum between about700 nm to about 775 nm.

Clause 29. The isolated polynucleotide of clause 25, wherein the CBRvariant polypeptide emits light having a spectral maximum of about 725nm.

Clause 30. The isolated polynucleotide any one of the preceding clauses,wherein the CBR variant polypeptide has altered substrate specificitycompared to a CBR polypeptide of SEQ ID NO: 1.

Clause 31. The isolated polynucleotide of clause 30, wherein the CBRvariant polypeptide has a change in relative specificity relative to theCBR variant polypeptide in the presence of a luciferin compared to aluciferin derivative.

Clause 32. The isolated polynucleotide of clause 30, wherein the CBRvariant polypeptide has a change in relative specificity relative to theCBR variant polypeptide in the presence of a luciferin derivativecompared to a different luciferin derivative.

Clause 33. The isolated polynucleotide of clause 31 or 32, wherein theluciferin derivative comprises:

Clause 34. The isolated polynucleotide of any one of the precedingclauses, wherein the variant CBR polypeptide has luciferase activity.

Clause 35. The isolated polynucleotide of any one of the precedingclauses, wherein the variant CBR polypeptide has a Km for PBI-4813 of atleast about 0.01 μM to at least about 5.00 μM.

Clause 36. The isolated polynucleotide of clause 35, wherein the variantCBR polypeptide has a Km for PBI-4813 of at least about 0.50 μM to atleast about 3.00 μM.

Clause 37. The isolated polynucleotide of clause 35, wherein the variantCBR polypeptide has a Km for PBI-4813 of at least about 0.82 μM or 2.41μM.

Clause 38. The isolated polynucleotide of any one of the precedingclauses, wherein the variant CBR polypeptide has a Km for PBI-4739 of atleast about 0.01 μM to at least about 5.00 μM.

Clause 39. The isolated polynucleotide of clause 38, wherein the variantCBR polypeptide has a Km for PBI-4739 of at least about 1.50 μM to atleast about 4.50 μM.

Clause 40. The isolated polynucleotide of clause 38, wherein the variantCBR polypeptide has a Km for PBI-4739 of at least about 2.33 μM or 3.95μM.

Clause 41. The isolated polynucleotide of any one of the precedingclauses, wherein the variant CBR polypeptide has a relative Vmax that isat least 2 fold higher than the relative Vmax of a CBR polypeptide ofSEQ ID NO: 1 using PBI-4813 as a substrate.

Clause 42. The isolated polynucleotide of any one of the precedingclauses, wherein the variant CBR polypeptide has a relative Vmax that isat least 2 fold higher than the relative Vmax of a CBR polypeptide ofSEQ ID NO: 1 using PBI-4739 as a substrate.

Clause 43. The isolated polynucleotide of any one of the precedingclauses, wherein the sequence has been codon-optimized.

Clause 44. The isolated polynucleotide of any one of the precedingclauses, wherein the sequence comprises a polynucleotide of SEQ ID NOs:6-9.

Clause 45. The isolated polynucleotide of any one of the precedingclauses, wherein the polynucleotide further encodes a polypeptide ofinterest linked to the CBR variant polypeptide, the polypeptide ofinterest and the CBR variant polypeptide capable of being expressed as afusion protein.

Clause 46. The isolated polynucleotide of clause 45, wherein thepolypeptide of interest comprises HALOTAG®.

Clause 47. A vector comprising the polynucleotide, or a fragmentthereof, of any one of the preceding clauses.

Clause 48. The vector of clause 47, wherein the polynucleotide isoperably linked to a promoter.

Clause 49. A cell comprising the polynucleotide of any one of clauses1-46 or the vector of clause 47 or 48.

Clause 50. A non-human transgenic animal comprising the cell of clause49.

Clause 51. A non-human transgenic animal comprising the polynucleotideof any one of clauses 1-46 or the vector of clause 47 or 48.

Clause 52. A CBR variant polypeptide encoded by the polynucleotide ofany one clauses 1-46.

Clause 53. A circularly permuted luciferase comprising the polypeptideencoded by the polynucleotide of any of clauses 1-46 or a fragmentthereof.

Clause 54. A fusion protein comprising a CBR variant polypeptide encodedby the polynucleotide of any one of clauses 1-46.

Clause 55. A near-infrared bioluminescence system comprising thepolynucleotide of any one of clauses 1-46 and a luciferin derivative.

Clause 56. The near-infrared bioluminescence system of clause 55,wherein the luciferin derivative comprises:

Clause 57. A method of producing a CBR variant polypeptide comprisinggrowing the cell of clause 47 under conditions that permit expression ofthe CBR variant polypeptide.

Clause 58. A method of producing a CBR variant polypeptide comprisingintroducing the vector of clause 47 or 48 into a cell under conditionswhich permit expression of the CBR variant polypeptide.

Clause 59. A kit comprising the polynucleotide of any one of clauses1-46 or the vector of clause 47 or 48.

Clause 60. A kit comprising the CBR variant polypeptide of clause 46.

Clause 61. The kit of clause 59 or 60 further comprising at least oneof:

(a)

and

(b) a buffer reagent.

Clause 62. A bioluminescence resonance energy transfer (BRET) systemcomprising: a first fusion protein including a first target protein anda bioluminescence donor molecule, wherein the bioluminescence donormolecule is a CBR variant encoded by the polynucleotide of any one ofclauses 1-46; a second fusion protein including a second target proteinand a fluorescent acceptor molecule; and a CBR substrate.

Clause 63. The BRET system of clause 62, wherein the CBR substrate is aluciferin or luciferin derivative.

Clause 64. The BRET system of clause 63, wherein the luciferinderivative comprises:

Clause 65. A method for measuring bioluminescence using at least one ofthe polynucleotide of any one of clauses 1-46; the vector of any one ofclauses 47 or 48; the cell of clause 49; the animal of any of clauses 50or 51; the CBR variant polypeptide of clause 52; the circularly permutedluciferase of clause 53; the fusion protein of clause 54, or thenear-infrared bioluminescence system of clause 55 or 56.

Clause 66. The method of clause 65, wherein the bioluminescence ismeasured in a live, intact non-human animal.

Clause 67. A method of measuring the enzymatic activity of a luminogenicprotein, the method comprising: contacting a luminogenic protein, adeprotecting enzyme, and a protected luminophore; and detecting lightproduced from the composition, wherein the luminogenic protein is a CBRvariant encoded by the polynucleotide of any one of clauses 1-46 and theluminophore is a luciferin derivative comprising:

Clause 68. The method of clause 67, wherein the enzymatic activity ismeasured in a live, intact non-human animal.

Clause 69. A method for measuring the activity of a non-luminescentenzyme of interest, the method comprising: (a) providing a luminogenicmolecule wherein the molecule is a substrate for the non-luminescentenzyme of interest and a pro-substrate of a CBR variant encoded by thepolynucleotide of any one of clauses 1-46; (b) contacting theluminogenic molecule with at least one non-luminescent enzyme ofinterest and at least one CBR variant to produce a reaction mixture; and(c) determining activity of the non-luminescent enzyme of interest bymeasuring luminescence of the reaction mixture.

Clause 70. The method of clause 69, wherein the luminogenic molecule isa modification of

Clause 71. The method of any one of clauses 69 and 70 wherein thenon-luminescent enzyme of interest is a protease enzyme, a cytochromeP450 enzyme, a monoamine oxidase enzyme, or a glutathione S-transferaseenzyme.

Clause 72. The method of any one of clauses 69 to 71 wherein theactivity of the non-luminescent enzyme is measured in a live, intactanimal.

Clause 73. A method to detect the presence of at least two molecules ina sample or a cell, the method comprising: contacting the sample or cellwith a first reporter molecule comprising a CBR variant encoded by thepolynucleotide of any one of clauses 1-46, wherein the first reportermolecule is operatively linked to a first component of the sample orcell; contacting the sample with a second reporter molecule, wherein thesecond reporter molecule is operatively linked to a second component ofthe sample or cell; and detecting the presence of the first and secondreporter molecules to determine the presence and/or amounts of the firstand second components in the sample or cell.

Clause 74. A method to detect an interaction between a first protein anda second protein in a sample, the method comprising:

(a) contacting a sample with:

wherein the sample comprises:

-   -   (i) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises a first fragment of a        luminescent enzyme and a first protein; and    -   (ii) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a second fragment of        the luminescent enzyme and a second protein; and

(b) detecting luminescence in the sample,

wherein the detection of luminescence indicates an interaction betweenthe first protein and the second protein, wherein the luminescent enzymeis encoded by the isolated polynucleotide of any one of clauses 1-46.

Clause 75. The method of clause 74, wherein when the first protein andsecond protein interact, the first fragment of the luminescent enzymeand the second fragment of the luminescent enzyme reconstitute afull-length enzyme capable of stably binding the cell-permeablesubstrate.

Clause 76. A method to detect an interaction between a first protein anda second protein in a sample, the method comprising:

(a) contacting a sample with:

wherein the sample comprises:

-   -   (i) a first polynucleotide encoding a first fusion protein,        wherein the first fusion protein comprises a luminescent enzyme        and a first protein, wherein the luminescent enzyme is encoded        by the isolated polynucleotide of any one of clauses 1-46; and    -   (ii) a second polynucleotide encoding a second fusion protein,        wherein the second fusion protein comprises a fluorescent        acceptor molecule and a second protein; and

(b) detecting bioluminescence resonance energy transfer (BRET) in thesample, indicating an interaction or close proximity of thebioluminescent donor and the fluorescence acceptor.

We claim:
 1. An isolated polynucleotide encoding a click beetle redluciferase (CBR) variant polypeptide having at least 80% amino acidsequence identity to SEQ ID NO: 1 and comprising an amino acidsubstitution at a position corresponding to positions 389, 444, and 251of SEQ ID NO: 1, or an amino acid substitution at a positioncorresponding to positions 334 and 351 of SEQ ID NO: 1, wherein thevariant CBR polypeptide has at least one of enhanced luminescence,altered light emission wavelength, altered substrate specificity, or acombination thereof, as compared to a CBR polypeptide of SEQ ID NO: 1.2. The isolated polynucleotide of claim 1, wherein the CBR variantpolypeptide further comprises an amino acid substitution at a positioncorresponding to positions 51 and 444 of SEQ ID NO:1.
 3. The isolatedpolynucleotide of claim 2, wherein the amino acid substitutions compriseS51N and S444R.
 4. The isolated polynucleotide of claim 1, wherein theCBR variant polypeptide further comprises a substitution correspondingto at least one of R4H, H16Q, H34Y, D47E, S51N, Y52C, F55L/V, K72E,179V, M73K/T, N74S, E82G, N83H, F87S, I89V, V104D, I109N/V, L113Q,M117T, I119F/T, I124V, N130K, 1131N/T, N133D, L136N, F144L, K146E,N156D, N156K, G159D, Y170C, K179S, V186A, H218L/Y, G225S,T226C/G/H/N/Q/Y, L228P, I229V, V234A, Y252C, V255D/F, F253K, R280S,S281N/Q, V285A, L309T, E319G, N329D, C3353, F337E, I346N, Q348H/E,L350P, D352N, K355G, S358P, T363A/S, I370T, K3901, M393K/L, V394M,N400D, N401S, I409T, D412G, Y422C, V431A, E437G, I439V, Q445H, E453K,L455D, K457N, D471V, E473A, S479T, K484E/M/R, E489V, Y496H, E501G,V503M, Y508C, V516A, T528A, E531G, Q535H, L537W, K539R, or combinationsthereof, of SEQ ID NO:
 1. 5. The isolated polynucleotide of claim 1,wherein the amino acid substitutions comprise 1389F, S444R, and G251S.6. The isolated polynucleotide of claim 1, wherein the amino acidsubstitutions comprise R334S and G351R.
 7. The isolated polynucleotideof claim 1, wherein the sequence has been codon-optimized.
 8. Theisolated polynucleotide of claim 1, wherein the polynucleotide furtherencodes a polypeptide of interest linked to the CBR variant polypeptide,the polypeptide of interest and the CBR variant polypeptide capable ofbeing expressed as a fusion protein.
 9. An isolated polynucleotideencoding a click beetle red luciferase (CBR) variant polypeptidecomprising a polypeptide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQID NO: 4, wherein the variant CBR polypeptide has at least one ofenhanced luminescence, altered light emission wavelength, alteredsubstrate specificity, or a combination thereof, as compared to a CBRpolypeptide of SEQ ID NO:
 1. 10. A vector comprising the polynucleotideof claim
 1. 11. The vector of claim 10, wherein the polynucleotide isoperably linked to a promoter.
 12. A cell comprising the polynucleotideof claim
 1. 13. A kit comprising the polynucleotide of claim
 1. 14. Thekit of claim 13 further comprising at least one of: (a)

and (b) a buffer reagent.
 15. A CBR variant polypeptide encoded by thepolynucleotide of claim
 1. 16. A circularly permuted luciferasecomprising the CBR variant polypeptide of claim
 15. 17. A fusion proteincomprising a CBR variant polypeptide encoded by the polynucleotide ofclaim
 1. 18. A near-infrared bioluminescence system comprising the CBRvariant polypeptide of claim 15 and a luciferin derivative.
 19. A kitcomprising the CBR variant polypeptide of claim 15 and at least one of:(a)

and (b) a buffer reagent.